Practicability Reliability Intralab/Interlab QC Interlab/External QC QC materials Bovine control materials Matrix effect Precision study Nonlab. personnel SD CV Variance Inferential statistics T-test F-test Cumulative Sum Graph (CUSUM) Youden/Twin Plot Shewhart Levey-Jennings Chart Trend Shift Outliers Kurtosis Precision Accuracy Random error (Imprecision; Indeterminate) Systematic error (Inaccuracy/Determinate) MUST TO KNOW IN CLINICAL CHEMISTRY (From CC by Rodriguez) Quality Control Method is easily repeated Maintain accuracy and precision Daily monitoring of accuracy and precision Proficiency testing (Reference lab) Long-term accuracy Difference of >2: not in agreement w/ other lab Available for a min. of 1 yr Preferred (Human: biohazard) Not for immunochem, dye-binding and bilirubin Improper product manufacturing Unpurified analyte Altered protein First step in method evaluation 29% of errors (lab results) Dispersion of values from the mean Index of precision Relative magnitude of variability (%) SD2 Measure of variability Compare means or SD of 2 groups of data Means of 2 groups of data SD of 2 groups of data V-mask Earliest indication of systematic errors (trend) Compare results obtained from diff. lab Graphic representation of the acceptable limits of variation Gradual loss of reliability Cause: Deterioration of reagents (Systematic error) Values: one side or either side of the mean Cause: Improper calibration (Systematic error) Values: far from the main set of values Highly deviating values Random or systematic errors Degree of flatness or sharpness Random error Systematic error Causes: -Mislabeling -Pipetting error -Improper mixing of sample and reagents -Voltage/Temperature fluctuation -Dirty optics Parameters: SD and CV Causes: -Improper calibration -Deterioration of reagents -Contaminated solution -Sample instability/unstable reagent blanks lec.mt 04 |Page | 1 Multirule Shewhart procedure Test method Reference method Analytical Run Physiologic Limit POCT Quality Assurance Quality Patient Care Reference Range/ Interval Range/ Reference Values Wavelength Spectrophotometric meas. Photometric measurement LASER Visible region UV IR Stray light Diffraction gratings Prisms Nickel sulfate Cutoff filter Bandpass Alumina silica glass cuvet Quartz/plastic cuvet Borosilicate glass cuvet Photodetector Barrier layer cell/ photocell/ photovoltaic cell Phototube Photomultiplier tube -Diminishing lamp power -Incorrect sample and reagent volume Parameter: Mean Control rules + Control chart Westgard: at least 40 samples Westgard: preferably 100 samples Control and patient specimens assayed, evaluated, and report together Referred to as absurd value Performed by nonlab personnel Tripod: Program development Assessment and monitoring Quality improvement Test request forms, clear instruction for patient prep., specimen handling… At least 120 individuals should be tested in each age and sex category Analytical Methods Distance bet 2 successive peaks (nm) Lower frequency = Longer wavelength (Ex. Red) Higher frequency = Shorter wavelength (Ex. Violet) Meas. light intensity in a narrower wavelength Meas. light intensity w/o consideration of wavelength Multiple wavelength (uses filter only) Light Amplification by Stimulated Emission of Radiation Light source for spectrophotometry Tungsten light bulb Mercury arc Deuterium lamp Mercury arc Xenon lamp Hydrogen lamp Merst glower Globar (Silicone carbide) Wavelength outside the band Most common cause of loss of linearity Most commonly used monochromator Cutting grooves Rotatable Prevents stray light Anti-stray light ½ peak transmittance Most commonly used cuvet UV Strong bases Converts transmitted light into photoelectric energy Simplest detector No external voltage For filter photometers Contains anode and cathode Req external voltage Most common type lec.mt 04 |Page | 2 Galvanometer/Ammeter Absorbance Double beam spectro. Double-beam in space Double-beam in time Dydimium filter Holmium oxide filter Reagent blank Sample blank FEP Cesium and Lithium Lithium AAS Atomizer (nebulizer) Chopper Lanthanum/Strontium chloride Volumetric (Titrimetric) Turbidimetry Nephelometry Electrophoresis Iontophoresis Zone electrophoresis Endosmosis Cellulose acetate Agarose gel Polyacrylamide gel Electrophoretic mobility Most sensitive UV and visible region Meter or read-out device A = abc (a = absorptivity; b = length of light (1cm); c = concentration) A = 2 – log%T Splits monochromatic light into two components: One beam sample One beam reference soln or blank (corrects for variation in light source intensity) 2 photodetectors (sample beam and reference beam) 1 photodetector Monochromatic light sample cuvet and reference cuvet 600 nm 360 nm Color of reagents Optical interference (Hgb) Meas. light emitted by a single atom burned in a flame Principle: Excitation Lt. source and cuvette: Flame For excited ions (Na+, K+) Internal standards (FEP) Correct variations in flame Preferred internal std Potent antidepressant Meas. light absorbed by atoms dissociated by heat Principle: Dissociation (unionized, unexcited, ground state) Lt. source: Hollow-cathode lamp For unexcited trace metals (Ca++ and Mg++) More sensitive than FEP Convert ions atoms Modulate the light source Complex with phosphate Avoid calcium interference Unknown sample is made to react with a known solution in the presence of an indicator Light blocked Meas. abundant large particles (Proteins) Depend on specimen concentration and particle size Meas. amt of Ag-Ab complexes Scattered light Depends on wavelength and particle size Migration of charged particles in an electric field Migration of small charged ions Migration of charged macromolecules Movement of buffer ions and solvent relative to the fixed support Ex: gamma globulins Molecular size Electrical charge Charge and molecular size 20 fractions (ex. isoenzymes) Directly proportional to net charge Inversely proportional to molecular size & viscosity of the supporting medium lec.mt 04 |Page | 3 Isoelectric focusing Densitometry Capillary electrophoresis Southern blot Northern blot Western blot Chromatography Paper chromatography TLC Retention factor (Rf) value Gas chromatography Gas Solid chromatography Gas Liquid chromatography Mass Spectrometry GC-MS MS/MS HPLC Hydrophilic gel Hydrophobic gel Ion exchange chromatography Partition chromatography Affinity chromatography Adsorption chromatography Fluorometry/Molecular Luminescence Spectro. Quenching Molecules migrate through a pH gradient pH = pI For isoenzymes: same size, different charge Scan & quantitate electrophoretic pattern Electro-osmotic flow DNA RNA Proteins Separation by specific differences in physical-chemical characteristics of the different constituents Fractionation of sugar and amino acid Sorbent: Whatman paper Screening: Drugs Relative distance of migration from the point of application Rf = Distance leading edge of component moves Total distance solvent front moves Separation of steroids, barbiturates, blood, alcohol, and lipids Volatile compounds Specimens vaporized Mobile phase: Inert gases Differences in absorption at the solid phase surfaces Differences in solute partitioning between the gaseous mobile phase and the liquid stationary phase Fragmentation and ionization Gold standard for drug testing Detect 20 inborn errors of metabolism from a single blood spot Most widely used liquid chromatography Fractionation of drugs, hormones, lipids, carbohydrates and proteins Gel filtration Separation of enzymes, antibodies and proteins Ex: Dextran and agarose Gel permeation Separation of triglyceride and fatty acid Ex: Sephadex Separation depends on the sign and ionic charge density Based on relative solubility in an organic solvent (nonpolar) and an aqueous solvent (polar) For lipoproteins, CHO and glycated hemoglobins Based on differences between the adsorption and desorption of solutes at the surfaces of a solid particle Det. amt. of lt. emitted by a molecule after excitation by electromagnetic radiation Lt. sources: Mercury arc and Xenon lamp (UV) Lt. detector: Photomultiplier tubes 2 monochromators: Primary filter – selects wavelength absorbed by the solution to be measured Secondary filter – prevents incident light from striking the photodetector Sensitivity: 1000x than spectro Major disadvantage of fluorometry pH and temperature changes, chemical contaminants, UVL changes lec.mt 04 |Page | 4 Borosilicate glasswares Boron-free/Soft glasswares Corex (Corning) Vycor (Corning) Flint glass TD: To deliver TC: To contain Blowout Self-draining Transfer pipet Graduated or measuring pipet Micropipettes Air displacement pipet Positive displacement pipet Dispenser/Dilutor pipet Distilled H2O Mercury Acid dichromate (H2SO4 + K2Cr2O4) Continuous flow analyzer Centrifugal analyzer Instrumentation For heating and sterilization Ex: Pyrex and Kimax High resistance to alkali Special alumina-silicate glass Strengthened chemically than thermally 6x stronger than borosilicate For high thermal, drastic heat and shock Can be heated to 900OC Soda-lime glass + Calcium, Silicon, Sodium oxides Easy to melt For making disposable glasswares Exact amount Does not disperse the exact volume w/ etched rings on top of pipet w/ o etched rings Drain by gravity Volumetric: for non-viscous fluid; self-draining Ostwald folin: for viscous fluid; w/ etched ring Pasteur: w/o consideration of a specific volume Automatic macro-/micropipets Serological: w/ graduations to the tip (blowout) Mohr: w/o graduations to the tip (self-draining) Bacteriologic Ball, Kolmer and Kahn Micropipettes: <1 mL TC pipets: Sahli-Hellige pipet Lang-Levy pipet RBC and WBC pipets Kirk and Overflow pipets Piston: suction Disposable tip Piston barrel (like a hypodermic syringe) Liquid: common reservoir dispense repeatedly Calibrating medium for TD pipettes Calibrating medium for TC pipettes Cleaning solution for glasswares Common reaction vessel Air bubbles: separates and cleans Glass coil: mix Examples: “STS” Simultaneous Multiple Analyzer (SMA) Technicon Autoanalyzer II SMAC Acceleration and deceleration of the rotor Advantage: Batch analysis Examples: “RICC” Cobas-Bio (Roche) IL Monarch CentrifiChem lec.mt 04 |Page | 5 Discrete Analyzer Thin-Film Analyzers (Dry slide technology) Carry over Batch testing Parallel testing Random access testing Sequential testing Open reagent system Closed reagent system Exercise RotoChem Most popular Req. vol: 2-6 μL Uses positive-displacement pipets Run multiple-tests-one-sample-at-a-time Random access capability (STAT) Examples: Vitros Dimension Dade Beckman ASTRA System (4 & 8) Hitachi Bayer Advia Roche Cobas Integra 800 Roche Analytics P Module Automated Clinical Analyzer (ACA) Star (Dade) Dupont ACA Abbott ABA-100 Bichromatic Analyzer ABA-200 VP Analyzer American Monitor KDA Olympus Demand 4 or 5 layers: -Spreading layer -Scavenger layer - Ascorbate Oxidase -Reagent layer -Indicator layer -Support layer Colored reaction Reflectance spectrophotometry Examples: “KV2(75)” Kodak Ektachem Vitros 750XRC Vitros 550XRC Transport of quantity of analyte or rgt from one specimen rxn into another, and contaminating a subsequent one All samples loaded at the same time Single test is conducted on each sample One specimen More than one test is analyzed Any sample Any test Any sequence STAT Multiple tests analyzed one after another on a given specimen System other than manufacturer’s reagents can be utilized for measurement The operator can only use the manufacturer’s reagents Patient Preparation Increased: GU2FT C2L3A5P2 GH Urea Urinary protein (Proteinuria) Fatty acid Testosterone lec.mt 04 |Page | 6 Fist clenching Fasting Basal state collection Diet Turbidity/Lactescence Icterisia Icteric samples Upright/supine (lying) position Supine Sitting/Standing Sitting Supine CPK (muscle) Creatinine (muscle) Lactate LH LD (muscle) ACP Aldolase (muscle) AST ALT Ammonia Pyruvate Prolactin Decreased: Glucose Increased: “LPP” Lactate Potassium Phosphate 8-16 hours: Glucose Lipids Lipoproteins Increased: Bilirubin (48 hours) Triglyceride (72 hours) Glucose Cholesterol Triglyceride Electrolytes Increased: “GLUC2H” Glucose Lipids Urea (High protein diet) Caffeine: increases glucose Catecholamines 5-HIAA (From Serotonin) Triglyceride >400mg/dL Bilirubin: 25.2 mg/dL Interfere with: "TACGu” Total Protein Albumin Cholesterol Glucose Preferred position Patient should be seated/supine at least 20 mins before blood collection to prevent hemodilution or hemoconcentration Vasoconstriction Reduced plasma volume Increased: “ECA” Enzymes Calcium Albumin Hemoconcentration lec.mt 04 |Page | 7 Increased: “P(u)BLIC” Proteins BUN Lipids Iron Calcium Standing Supine Hemodilution Decreased: “TLC” Triglycerides Lipoproteins Cholesterol Prolonged standing Increased: K+ (muscles) Prolonged bedrest Decreased: Albumin (Fluid retention) Tourniquet Recommended: 1 minute application Prolonged tourniquet app. Hemoconcentration Anaerobiosis Increased: “C2LEA2K” Calcium Cholesterol Lactate Enzymes Ammonia Albumin K+ Tobacco smoking (Nicotine) Increased: “TUNG2C3” Triglycerides Urea Nonesterified fatty acid Glucose GH Catecholamines Cortisol Cholesterol Alcohol ingestion Increased: “THUG” Triglycerides Hypoglycemia (chronic alcoholism) Uric acid/Urates GGT Ammonia Increases by 100-200μg/L/cigar Stress (anxiety) Increased: “LAGIC” Lactate Albumin Glucose Insulin Cholesterol Drugs Medications affecting plasma volume can affect protein, BUN, iron, calcium Hepatotoxic drugs: increased liver function enzymes Diuretics: decreased sodium and potassium Diurnal variation "CA3PI2TG” Cortisol ACTH ACP lec.mt 04 |Page | 8 Sleeping patients Unconscious patients Venipuncture Tourniquet Needle After blood collection BP cuff as tourniquet Benzalkonium chloride (Zephiran) IV line on both arms IV fluid contamination Renin blood level Basal state collection Lancet Incision (Skin puncture) 1.5-2.4mm Arterialized capillary blood Flea Indwelling umbilical artery 1000-3000 RCF for 10 mins Hemolysis Aldosterone Prolactin Iron Insulin Thyroxine GH Specimen Collection and Handling Must be awakened before blood collection Ask nurse or relative Identification bracelet Median Cubital (1st) Cephalic (2nd) Basilic (3rd) Velcro or Seraket type 3-4 inches above the site Not exceed 1 minute Bevel up 15-30O angle Length: 1 or 1.5 inch (Butterfly needle: ½ to ¾ inch) Cotton site Apply pressure for 3-5 minutes Inflate to 60 mmHg Disinfectant for ethanol testing Dilution – 1:750 Discontinue IV for 2 minutes Collect sample below the IV site Initial sample (5mL) discard Increased: Glucose (10% contam. w/ 5% dextrose increased bld glucose by 500 mg/dL) Chloride Potassium Sodium Decreased: Urea Creatinine Collected after a 3-day diet, from a peripheral vein Early morning blood collection 12 hours after the last ingestion of food 1.75mm: preferred length to avoid penetrating the bone <2.0mm (infants and children) 2-3mm (adults) Distance from the skin surface to bone or cartilage (middle finger) Earlobe: Preferred site Lateral plantar heel surface: most commonly used site Minute metal filling which may be inserted into the capillary tube before collecting blood to help mix the specimen while the blood is entering the tube Best site for blood gas analysis (newborns) Centrifugation requirement Increased: “KLA6MP ITC2” -K+ -LDH (150x) -ACP -ALP lec.mt 04 |Page | 9 Refrigeration/Chilling (Low temp) Photosensitive analytes Oxalate Citrate EDTA Fluoride Heparin Lithium heparin Orange top tube Royal blue top tube Brown top tube Tan top tube Black top tube -Aldolase -ALT -AST -Albumin -Mg2+ -Phosphorus -Iron -Total protein Affects bilirubin levels Inhibits lipase Required for: “ABCGLRP2” Ammonia Blood gases Catecholamines Gastrin Lactic acid Renin PTH Pyruvate Decreased: LD 4 and 5 Increased: ALP Bilirubin Beta-carotene Folate Porphyrins Vitamins A and B6 Insoluble salt 1-2 mg/mL blood Non-ionized form 3.2-3.8 g/dL (1:9 ratio) Chelation 1-2 mg/mL blood Versene: disodium salt Sequestrene: Dipotassium salt Weakly dissociated calcium component 2 mg/mL blood: anti-glycolytic 10 mg/mL blood: anticoagulant A.k.a. Mucoitin polysulfuric acid Universal anticoagulant Antithrombin 0.2 mg/mL blood For glucose, BUN, ionized calcium, electrolyte studies (K+: best) and creatinine Additive: Thrombin Additives: None; Na2EDTA Sodium heparin Lead testing Lead testing Additive: Buffered sodium citrate lec.mt 04 |Page | 10 Respinning gel tubes Thixotropic gel % w/v % v/v % w/w Molarity Moles To prepare a molar solution To convert % w/v to Molarity Normality Equivalent weight (EW) To prepare a normal solution of solids To convert % w/v to Normality Normality Molarity Molality Milliequivalents Millimoles Ratio Dilution 0.179 0.01 2.27 Analytical reagent (AR) grade Ultrapure reagents Chemically Pure (CP) or For ESR Increases potassium Gel separator (SG: 1.04) Serum: (SG: 1.03) RBC: (SG: 1.05) Laboratory Mathematics Grams of solute = % solution desired x total volume desired 100 mL of solute = % solution desired x total volume desired 100 Grams of solute = % solution desired x grams of the total solution 100 M= _grams of solute_______ GMW x volume of solution Mol = weight (grams) GMW Grams of solute = Molarity x GMW of the solute x Volume (L) desired M = % w/v 10 GMW N = _Grams of solute_ EW x volume (L) EW = __MW___ valence Grams of solute = Normality x EW x Volume (L) N = w/v 10 EW N = Molarity x Valence M = Normality valence m = Grams of solute__ MW x kg of solvent mEq/L = mg/dL 10 valence MW mmol/L = mg/dL 10 MW Ratio = _Volume of solute_ Volume of solvent Dilution = __Volume of solute__ Volume of solution Conversion factor for iron (mg/dL μmol/L) Conversion factor for phospholipid (g/dL to g/L) Conversion factor for folate For qualitative and quantitative analyses For accuracy Established by American Chemical Society (ACS) Uses: Trace metal analysis and preparation of standard solutions Additional purification steps Ex: Spectrograde, nanograde, HPLC grade Uses: Chromatography, atomic absorption, immunoassays Indicates that the impurity limitations are not stated lec.mt 04 |Page | 11 Pure Grade Technical/Commercial grade United States Pharmacopoeia (USP) and National Formulery (NF) Preparation of reagent grade water Type I Rgt Water Type II Rgt Water Type III Distilled water Deionized water Occupational Safety and Health Act (OSHA) College of American Pathologists (CAP) Tests for water purity Detergent-contaminated water Hard water NCCLS Dilute solution Concentrated solution Saturated solution Super saturated solution Purity is delivered by meas. of melting point or boiling point In manufacturing Never used in clin. lab. testing For human consumption Not applicable for lab. analysis Purpose: For drug manufacturing Filtration (1st) Distillation, Ion exchange, Reverse Osmosis Min. interference Max. water purity Used immediately For ultramicrochemical analyses, measurements of nanogram or subnanogram concentrations, tissue or cell methods (microscopy) and preparation of standard solutions Uses: FEP, AAS, blood gases and pH, enzyme studies, electrolyte testing, HPLC, trace metal and iron studies For clinical laboratory use (hematology, microbiology, immunology, chemistry) For prep. of rgts and QC materials For washing glasswares For urinalysis, parasitology and histology Purified to remove almost all organic materials Free from mineral salts; removed by ion exchange processes Organic material may still be present Req. manuf. to indicate lot no., physical or biological health hazard of the chem.. rgts, and precautions for safe use and storage Recommends that a lab. document culture growth, pH and specific water resistance on reagent grade water Microbiological content pH Resistivity Chemical oxygen demand Ammonia Ions Metals Alkaline pH Contains calcium, iron and other dissolved elements Now: Clinical and Laboratory Standards Institute (CLSI) Relatively little solute Large quantity of solute in solution Excess of undissolved solute particles Greater concentration of undissolved solute particles than does a saturated solution of the same substance Primary standard Highly purified (IUPAC) Measured directly to produce a substance of exact known concentration Secondary standard Low purity Concentration is determined by comparison w/ a primary standard Laboratory Safety National Fire Protection Association (NFPA) Classification of Fires Class A fire Ordinary combustibles: paper, cloth, rubbish, plastics, wood Extinguisher: Water (A), Dry chemical (ABC), loaded steam lec.mt 04 |Page | 12 Class B fire Flammable liquids: grease, gasoline, paints, oil Extinguisher: Dry chemical (ABC), carbon dioxide (BC), halon foam (BC) Class C fire Electrical equipment and motor switches Extinguisher: Dry chemical (ABC), Carbon dioxide (BC), halon (BC) Class D fire Flammable metals: mercury, magnesium, sodium, lithium Extinguisher: Metal X Fought be fire fighters only Class E fire Detonation (Arsenal fire) Allowed to burn out and nearby materials protected Standard Hazards Identification System (Diamond-shaped color coded symbol) Blue quadrant Health hazard Red quadrant Flammable hazard Yellow quadrant Reactivity/Stability hazard White quadrant Other special information Chemical spills 1st step: assist/evacuate personnel 1:10 dilution of chlorine To disinfect and clean bench tops bleach (10%) In contact with the area for at least 20 minutes HBV: 10 minutes HIV: 2 minutes Poisonous vapors Chloroform Methanol Carbon tetrachloride Bromide Ammonia Formaldehyde Mercury Flammable and Acetone combustible solvents Ethanol Toluene Methanol Xylene Benzene Isopropanol Heptane Flammable liquids Flash point below 37.8OC Combustible liquids Flash point at or above 37.8OC Strong acids or bases Neutralized before disposal Water should NEVER be added to concentrated acid Ether Deteriorate over time hazardous Forms explosive peroxides Benzidine Known carcinogen Fumehoods Ventilation: velocity of 100-120 ft/min Safety showers Deliver 30-50 gal/min of H2O at 20-50 psi Carbohydrates Glycol aldehyde The simplest carbohydrate Sucrose Most common nunreducing sugar Pancreas Exocrine: Enzymes (AMS, LPS) Endocrine: Hormones (Insulin, glucagon, somatostatin) Hyperglycemic Hormones “GAG CHET” Glucagon ACTH GH lec.mt 04 |Page | 13 Hyperglycemia (≥126 mg/dL) Hypoglycemia Whipple’s triad (Hypoglycemia) 6:1 Type 1 DM Complications of Type I DM Type 2 Gestational DM OGTT (GDM) Impaired fasting glucose (Pre-diabetes) Impaired glucose tolerance Cortisol Human Placental Lactogen Epinephrine Thyroxine Electrolyte Imbalance: Decreased: Sodium, Bicarbonate Increased: Potassium 50-55 mg/dL = Symptoms ≤50 mg/dL = Diagnostic Low blood glucose concentration Typical symptoms Symptoms alleviated by glucose administration Ratio of BHA to AA in severe DM (Normal = 1:1) IDDM Juvenile Onset Brittle Ketosis-prone 80-90% reduction of beta-cells Symptomatic Type 1 DM HLA-DR3 and DR4 (+) Glutamic acid decarboxylase (GAD65) (+) Insulin autoantibodies (+) Microalbuminuria: 50-200 mg/24 hours = Diabetic nephropathy (-) C-peptide Microvascular disorders: Nephropathy Neuropathy Retinopathy NIDDM Adult type/Maturity Onset Stable Ketosis-resistant Receptor-deficient Insulin resistance: relative insulin deficiency Strong genetic predisposition Geneticist’s nightmare If untreated glucose: >500 mg/dL nonketotic hyperosmolar coma Screening: 1hr GCT (50g) – bet. 24 and 28 weeks of gestation Confirmatory: 3-hr GTT (100g) Infants: at risk for respiratory distress syndrome, hypocalcemia, hyperbilirubinemia After giving birth, evaluate 6-12 weeks postpartum Converts to DM w/in 10 years in 30-40% of cases FBS = ≥95 mg/dL 1-Hr = ≥ 180 mg/dL 2-Hr = ≥ 155 mg/dL 3-Hr = ≥ 140 mg/dL GDM = 2 plasma values of the above glucose levels are exceeded FBS = 100-125 mg/dL FBS = <126 mg/dL 2-Hr OGTT = 140-199 mg/dL lec.mt 04 |Page | 14 FBS CSF glucose Peritoneal fluid glucose Plasma glucose increases w/ age w/in 1 hour (Preferably w/in 30 mins) 5-7%/hr 1-2 mg%/hr Copper reduction methods Folin Wu Nelson-Somogyi Neocuproine method Benedict’s method Alkaline Ferric Reduction method (Hagedorn-Jensen) Ortho-toluidine (Dubowski method) Glucose oxidase Mutarotase NADH/NADPH Polarographic glucose oxidase Hexokinase method G-6-PD Interfering substances (Glucose oxidase) Hemolysis (>0.5 g/dL Hgb) Dextrostics OGTT IVGTT Requirements for OGTT Glucose load HbA1c WB = 15% lower than in serum or plasma VB = 7 mg/dL lower than capillary and arterial blood 60-70% of the plasma glucose Same with plasma glucose Fasting: 2 mg/dL/decade Postprandial: 4 mg/dL/decade Glucose challenge: 8-13 mg/dL/decade Separate serum/plasma from the cells Glycolysis at room temperature Glycolysis at refrigerated temperature Cupric Cuprous Cuprous oxide Cuprous ions + phosphomolybdate phosphomolybdenum blue Cuprous ions + arsenomolybdate arsenomolybdenum blue Cuprous ions + neocuproine Cuprous-neocuproine complex (yellow) Reducing substances in blood and urine Ferricyanide ---(Glucose)--> Ferrocyanide (Yellow) (Colorless) Schiff’s base Measures beta-D-glucose (65%) Converts alpha-D-glucose (35%) to beta-D-glucose (65%) Absorbance at 340nm Consumption of oxygen on an oxygen-sensing electrode O2 consumption α glucose concentration Most specific method Reference method Uses G-6-PD Most specific enzyme rgt for glucose testing False-decreased Bilirubin Uric acid Ascorbate Major interfering substance in hexokinase method (false-decreased) Cellular strip Strip w/ glucose oxidase, peroxidase and chromogen Janney-Isaacson method (Single dose) = most common Exton Rose (Double dose) Drink the glucose load within 5 mins For patients with gastrointestinal disorders (malabsorption) Glucose: 0.5 g/kg body weight Given w/in 3 mins 1st blood collection: after 5 mins of IV glucose Ambulatory Fasting: 8-14 hours Unrestricted diet of 150g CHO/day for 3 days Do not smoke or drink alcohol 75 g = adult (WHO std) 100 g = pregnant 1.75 g glucose/kg BW = children 2-3 months Glucose = beta-chain of HbA1 lec.mt 04 |Page | 15 IDA and older RBCs RBC lifespan disorders Fructosamine (Glycosylated albumin/ plasma protein ketoamine) Galactosemia Essential fructosuria Hereditary fructose intolerance Fructose-1,6-biphosphate deficiency Glycogen Storage Disease Ia = Von Gierke II = Pompe III = Cori Forbes IV = Andersen V = McArdle VI = Hers VII = Tarui XII = Fanconi-Bickel CSF glucose < 0.5 C-peptide 5:1 to 15:1 D-xylose absorption test Gerhardt’s ferric chloride test Nitroprusside test Acetest tablets Ketostix KetoSite assay Normal Values (Carbohydrates) 1% increase in HbA1c = 35 mg/dL increase in plasma glucose 18-20% = prolonged hyperglycemia 7% = cutoff Specimen: EDTA whole blood Test: Affinity chromatography (preferred) High HbA1c Low HbA1c 2-3 weeks Useful for patients w/ hemolytic anemias and Hgb variants Not used in cases of low albumin Specimen: Serum Congenital deficiency of 1 of 3 enzymes in galactose metabolism Galactose-1-phosphate uridyl transferase (most common) Galactokinase Uridine diphosphate galactose-4-epimerase Autosomal recessive Fructokinase deficiency Defective fructose-1,6-biphosphate aldolase B activity Failure of hepatic glucose generation by gluconeogenic precursors such as lactate and glycerol Autosomal recessive Defective glycogen metabolism Test: IVGTT (Type I GSD) Glucose-6-Phosphatase deficiency (most common worldwide) Alpha-1,4-glucosidase deficiency (most common in the Philippines) Debrancher enzyme deficiency Brancher enzyme deficiency Muscle phosphorylase deficiency Liver phosphorylase deficiency Phosphofructokinase deficiency Glucose transporter 2 deficiency Collect blood glucose at least 60 mins (to 2 hrs) before the lumbar puncture (Because of the lag in CSF glucose equilibrium time) Normal CSF : serum glucose ratio Formed during conversion of pro-insulin to insulin Normal C-peptide : insulin ratio Differentiate pancreatic insufficiency from malabsorption (low blood or urine xylose) Acetoacetate 10x more sensitive to acetoacetate than to acetone Acetoacetate and acetone Detects acetoacetate better than acetone Detects beta-hydroxybutyrate but not widely used RBS = <140 mg/dL FBS = 70-100 mg/dL HbA1c = 3-6% Fructosamine = 205-285 μmol/L 2-Hr PPBS = <140 mg/dL GTT: 30 mins = 30-60 mg/dL above fasting lec.mt 04 |Page | 16 Phospholipids Sphingomyelin Forms of phospholipids TLC + Densitometric quantitation Microviscosity Cholesterol LCAT Apo A-1 Cholesterol increases after the age of 50 Liebermann Burchardt Salkowski Color developer mixture (Cholesterol) One-step method Two-step method Three-step method Four-step method Abell, Levy and Brodie mtd (Chemical method) Triglycerides Triglyceride increases after the age of 50 Van Handel & Zilversmith (Colorimetric) Hantzsch Condensation (Fluorometric) Modified Van Handel and Zilversmith (Chemical method) 1-Hr = 20-50 mg/dL above fasting 2-Hr = 5-15 mg/dL above fasting 3-Hr = fasting level or below Lipids Most abundant lipid Amphipathic: polar (hydrophilic head) and nonpolar (hydrophobic side chain) Reference material during 3rd trimester of pregnancy Concentration is constant as opposed to lecithin Not derived from glycerol but from sphingosine (amino alcohol) 70% Lecithin/Phosphatidyl choline 20% Sphingomyelin 10% Cephalin Method for L/S ratio Measured by fluorescence polarization Not a source of fuel Not affected by fasting 70% Cholesterol ester (plasma/serum) 30% Free cholesterol (plasma/serum and RBC) Esterification of cholesterol Activator of LCAT 2 mg/dL/year between 50 and 60 years old Cholestadienyl Monosulfonic acid Green end color Cholestadienyl Disulfonic acid Red end color Glacial acetic acid Acetic anhydride Conc. H2SO4 Colorimetry (Pearson, Stern and Mac Gavack) Color. + Extraction (Bloor’s) Color. + Extract. + Saponification (Abell-Kendall) Color. +Extract. + Sapon. + Precipitation (Schaenheimer Sperry, Parekh and Jung) CDC reference method for cholesterol: -Hydrolysis/saponification (Alc. KOH) -Hexane extraction -Colorimetry (Liebermann-Burchardt) Most insoluble lipid Main storage lipid in man (adipose tissue) – 95% Fasting: 12 hours 2 mg/dL/year between 50 and 60 years old Chromotropic acid (+) Blue color compound Diacetyl acetone (+) Diacetyl lutidine compound CDC reference method for triglycerides: -Alkaline hydrolysis -Chloroform extraction extract treated w/ silicic acid -Color reaction w/ chromotropic acid – meas. HCHO lec.mt 04 |Page | 17 Fatty acids Palmitic acid Stearic acid Oleic acid Linoleic acid Arachidonic acid Lipoprotein lipase (Lipemia clearing factor) Hepatic lipase Endothelial lipase Apolipoprotein Chylomicrons VLDL LDL HDL IDL Lp(a) (+) Pink colored Short chain = 4-6 C atoms Medium chain = 8-12 C atoms Long chain = >12 C atoms Saturated = w/o double bonds Unsaturated = w/ double bonds Substrate for gluconeogenesis Most is bound to albumin 16:0 18:0 18:1 18:2 20:4 Hydrolyzes TAG in lipoproteins, releasing fatty acid and glycerol Hydrolyzes TAG and phospholipids from HDL Hydrolyzes lipids on VLDL and IDL Hydrolyzes phospholipids and TAG in HDL Protein component of lipoprotein Amphipathic helix – ability of proteins to bind to lipids Largest and least dense Produced by the intestine SG: <0.95 80-95% TAG (exogenous) Apo B-48 (Major) EP: Origin Secreted by the liver SG: 0.95-1.006 65% TAG (endogenous) Apo B-100 (Major) EP: pre-beta Synthesized by the liver SG: 1.006-1.063 50% CE Apo B-100 (Major) EP: beta Cholesterol transport: LiverTissues Target of cholesterol lowering therapy Better marker for CHD risk Smallest but dense SG: 1.063-1.21 45-55% protein 26-32% phospholipid Apo A-1 (Major) EP: alpha Produced by the liver and intestine Reverse cholesterol transport: TissueLiver Product of VLDL catabolism Seen in Type 3 hyperlipoproteinemia (Apo E-III def.; beta-VLDL) SG: 1.006-1.019 Sinking pre-beta lipoprotein SG: 1.045-1.080 lec.mt 04 |Page | 18 LpX Beta-VLDL Lipoprotein methodologies Ultracentrifugation Electrophoresis Chemical precipitation 3-step procedure: Ultracentrifugation Precipitation Abell-Kendall assay Beta quantification + Ultracentrifugation + Chemical precipitation Immunoturbidimetric assay LDL Cholesterol Friedewald method De Long method Apo A-1 Apo B-100 Apo B-48 Apo C-II Apo D Apo E Apo(a) Apo B-100 EP: pre-beta (VLDL) UC: like LDL Independent risk factor for atherosclerosis Found in obstructive jaundice (cholestasis) and LCAT deficiency 90% FC and PL Apo C and albumin Floating beta-lipoprotein SG: <1.006 EP: beta (LDL) UC: like VLDL Found in type 3 hyperlipoproteinemia (Apo E-III def; IDL) Rich in cholesterol content than VLDL Specimen: sample from serum separator tubes (preferred) EDTA plasma: choice for research studies of LPP fractions Fasting state: TAG VLDL Nonfasting state: TAG CM Reference method for LPP quantitation Reagent: Potassium bromide (SG: 1.063) Ultracentrifugation of plasma for 24 hours Expressed in Svedberg units Electrophoretic pattern: (+) HDL VLDL LDL CM (Origin) (-) Agarose gel: sensitive medium VLDL: migrates w/ alpha2-globulin (pre-beta) Uses polyanions (heparin and divalent cations) and polyethylene glycol Dextran sulfate-Mg2+ Heparin-Mn2+ CDC Reference method for HDL Method for LDL Sample: EDTA plasma Measures Lipoprotein (a) Total Cholesterol – HDL – VLDL Most commonly used VLDL = TAG/2.175 (mmol/L) VLDL = TAG/5 (mg/dL) Not applicable if TAG is >400 mg/dL VLDL = TAG/2.825 (mmol/L) VDL = TAG/6.5 (mg/dL) Activates LCAT LDL LDL receptor CM (major) Not recognized by LDL receptor Activates LPL Activates LCAT Apo E-4: associated w/ high LDL, higher risk of CHD and Alzheimer’s disease Lp(a) Homologous to plasminogen lec.mt 04 |Page | 19 Abetalipoproteinemia (Basses-Kornzweig syn.) Niemann-Pick disease Tangier’s disease LPL deficiency (Chylomicronemia) LCAT deficiency Tay-Sachs disease Fredrickson Classification Type 1 Type 2a Type 2b Type 3 Type 4 Type 5 Normal Values (Lipids) Proteis Proteins Autosomal recessive Defective apo B synthesis Deficient fat soluble vitamins Sphingomyelinase deficiency Deficiency of HDL (1-2 mg/dL) Defects in the gene for the ABCA1 transporter TAG = 10,000 mg/dL Do not develop premature coronary disease (CM are not atherogenic) Abdominal pain and pancreatitis Fish-eye disease Low HDL Hexosaminidase A deficiency LPL deficiency (Chylomicronemia) Increased: CM (TAG) Familial hypercholesterolemia Increased: LDL (cholesterol) Combined hyperlipidemia (most common primary hyperlipidemia) Increased: LDL (cholesterol), VLDL (TAG) Dysbetalipoproteinemia Increased: IDL, (+) beta-VLDL (+) Apo E-II (+) Eruptive and palmar xanthomas Hypertriglyceridemia Increased: VLDL (TAG) Increased: VLDL (Endo.TAG), CM (Exo.TAG) Cholesterol: Desirable = <200 mg/dL Borderline high = 200-239 mg/dL High = >240 mg/dL Triglycerides: Desirable = <150 mg/dL Borderline high = 150-199 mg/dL High = 200-499 Very high = >500 mg/dL HDL: Low = <40 mg/dL (Cutoff) High = >60 mg/dL LDL: Optimal = <100 mg/dL Near/above optimal = 100-129 mg/dL Borderline high = 130-159 mg/dL High = 160-189 mg/dL Very high = >190 mg/dL Proteins First rank of importance Amphoteric: positive and negative charges Effective blood buffers Synthesized by the liver except immunoglobulins (plasma cells) Provide 12-20% of total daily body energy requirement Composed of 50-70% of the cell’s dry weight lec.mt 04 |Page | 20 Primary structure Secondary structure Tertiary structure Quarternary structure Albumin Glucogenic amino acids Ketogenic amino acids Simple proteins Conjugated proteins Nitrogen balance Negative nitrogen balance Positive nitrogen balance Prealbumin (Transthyretin) Albumin Alpha1-antitrypsin Alpha1-fetoprotein Alpha1-acid glycoprotein/ orosomucoid Alpha1-antichymotrypsin Haptoglobin (alpha2) Ceruloplasmin (alpha2) Amino acid sequence Det. the identity of protein, molecular structure, function binding capacity, recognition ability Winding of polypeptide chain Specific 3-D conformations: alpha-helix, beta-pleated sheath, bend form Actual 3-D configuration Folding pattern Physical and chemical properties of proteins Association of 2 or more polypeptide chains protein No quarternary structure Alanine (pyruvate) Arginine (alpha-ketoglutarate) Aspartate (oxaloacetate) Degraded to acetyl-CoA Leucine Lysine Hydrolysis Amino acids Fibrous: fibrinogen, troponins, collagen Globular: hemoglobin, plasma proteins, enzymes, peptide hormones Protein (apoprotein) + nonprotein moiety (prosthetic group) Metalloproteins: ferritin, ceruloplasmin, hemoglobin, flavoproteins Lipoproteins: VLDL, HDL, LDL, CM Glycoproteins: haptoglobin, alpha1-antitrypsin (10-40% CHO) Mucoproteins or proteoglycans: Mucin (CHO > CHON) Nucleoproteins: Chromatin (combined w/ nucleic acids) Balance bet. anabolism and catabolism Catabolism > anabolism Excessive tissue destruction Anabolism > catabolism Growth and repair processes Transports thyroxine and retinol (Vit. A) Landmark to confirm that the specimen is really CSF Maintains osmotic pressure Negative acute phase reactant Acute phase reactant Major inhibitor of protease activity 90% of alpha1-globulin band Gestational marker Tumor marker: hepatic and gonodal cancers Screening test for fetal conditions (Spx: maternal serum) Amniotic fluid: confirmatory test Increased: Hepatoma, spina bifida, neural tube defects Decreased: Down Syndrome (Trisomy 21) Low pI (2.7) Negatively charged even in acid solution Acute phase reactant Binds and inactivates PSA Increased: Alzheimer’s disease, AMI, infection, malignancy, burns Acute phase reactant Binds free hemoglobin (alpha chain) Copper binding (6-8 atoms of copper are attached to it) Has enzymatic activities lec.mt 04 |Page | 21 Alpha2-macroglobulin Group-specific component (Gc)-globulin (bet. alpha1 and alpha2) Hemopexin (beta) Beta2-microglobulin Transferrin/Siderophilin (beta) Complement (beta) Fibrinogen (bet. beta and gamma) CRP (gamma) Immunoglobulins (gamma) Myoglobin Troponins TnT (Tropomyosin-binding subunit) TnI (Inhibitory subunit or Actin-binding unit) TnC Glomerular proteinuria Tubular proteinuria Overload proteinuria Postrenal proteinuria Microalbuminuria CSF Oligoclonal banding Decreased: Wilson’s disease (copper skin, liver, brain, cornea [KayserFleisher rings]) Larges major nonimmunoglobulin protein Increased: Nephrotic syndrome (10x) Forms a complex w/ PSA Affinity w/ vitamin D and actin Binds free heme HLA Filtered by glomeruli but reabsorbed Negative acute phase reactant Major component of beta2-globulin fraction Pseudoparaproteinemia in severe IDA Increased: Hemochromatosis (bronze-skin), IDA C3: major Acute phase reactant Between beta and gamma globulins General scavenger molecule Undetectable in healthy individuals hsCRP: warning test to persons at risk of CAD Synthesized by the plasma cells IgG>IgA>IgM>IgD>IgE Marker: Ischemic muscle cells, chest pain (angina), AMI Most important marker for AMI Specific for heart muscle Det. unstable angina (angina at rest) Only found in the myocardium Greater cardiac specificity than TnT Highly specific for AMI 13x more abundant in the myocardium than CK-MB Very sensitive indicator of even minor amount of cardiac necrosis Binds calcium ions and regulate muscle contractions Most common and serious type Often called albuminuria Defective reabsorption Slightly increased albumin excretion Hemoglobinuria Myoglobinuria Bence-Jones proteinuria Urinary tract infection, bleeding, malignancy Type 1 DM Albumin excretion ≥30 mg/g creatinine (cutoff: DM) but ≤300 mg/g creatinine Microalbuminuria: 2 out of 3 specimens submitted are w/ abnormal findings (w/in 6 months) 2 or more IgG bands in the gamma region: Multiple sclerosis Encephalitis Neurosyphilis Guillain-Barre syndrome Neoplastic disorders lec.mt 04 |Page | 22 Serum Oligoclonal banding Alkaptonuria Homocystinuria MSUD PKU Normal Values (Proteins) Tests for GFR Tests for Renal Blood Flow Tests Measuring Tubular Function GFR Inulin clearance Creatinine clearance Urea clearance Cystatin C BUN 2.14 Fluoride or citrate Thiosemicarbazide Ferric ions Diacetyl monoxime method Urease method Leukemia Lymphoma Viral infections Ochronosis (tissue pigmentation) Impaired activity of cystathione beta-synthetase Elevated homocysteine and methionine in blood and urine Screen: Modified Guthrie test (Antagonist: L-methionine sulfoximine) Markedly reduced or absence of alpha-ketoacid decarboxylase 4 mg/dL of leucine is indicative of MSUD Screen: Modified Guthrie test (Antagonist: 4-azaleucine) Diagnostic: Amino acid analysis (HPLC) Deficiency of tetrahydrobiopterin (BH4) elevated blood phenylalanine Total protein = 6.5-8.3 g/dL Albumin = 3.5-5.0 g/dL Globulin = 2.3-3.5 g/dL Kidney Function Tests Clearance: -Inulin clearance -Creatinine clearance -Urea clearance Phenolsulfonphthalein dye test Cystatin C BUN Creatinine Uric acid Excretion: -Para-amino hippurate test (Diodrast test) -Phenolsulfonphthalein dye test Concentration: -Specific gravity -Osmolality Decreases by 1.0 mL/min/year after age 20-30 years 150 L of glomerular filtrate is produced daily Reference method Best alternative method Measure of the completeness of a 24-hour urine collection Excretion: 1.2-1.5 g creatinine/day Demonstrate progression of renal disease or response to therapy Low MW protease inhibitor FilteredNot secretedCompletely reabsorbed (PCT) Indirect estimates of GFR Its presence in urine denotes damage to PCT Synthesized from Ornithine or Kreb’s Henseleit cycle First metabolite to elevate in kidney diseases Better indicator of nitrogen intake and state of hydration BUN Urea (mg/dL) Inhibit urease Enhance color development (BUN mtd) Yellow diazine derivative Routinely used Urease: prepared from jack beans lec.mt 04 |Page | 23 Coupled urease Isotope dilution mass spectrometry NPN Creatinine Enzymatic methods (Creatinine) Direct Jaffe method Interferences (Direct Jaffe) Folin Wu Method Lloyd’s or Fuller’s Earth method Lloyd’s reagent Fuller’s earth reagent Jaffe reagent (Alk. picrate) Kinetic Jaffe method Azotemia Pre-renal azotemia Renal azotemia Post renal azotemia Uremia Urea ---(Urease)--> NH4 + Berthelot reagent (Measure ammonia) Glutamate dehydrogenase method UV enzymatic method Reference method For research purposes 45% Urea 20% Amino acid 20% Uric acid 5% Creatinine 1-2% Creatine 0.2% Ammonia Derived from alpha-methyl guanidoacetic acid (creatine) Produced by 3 amino acids (methionine, arginine, lysine) Most commonly used to monitor renal function Creatinine Aminohydrolase – CK method Creatinase-Hydrogen Peroxide method – benzoquinonemine dye (red) Creatininase (a.k.a. creatinine aminohydrolase) Formation of red tautomer of creatinine picrate Falsely increased: Ascorbate Glucose Uric acid Alpha-keto acids (+) Red orange tautomer True measure of creatinine Sensitive and specific Uses adsorbent to remove interferences (UA, Hgb, Bili) Sodium aluminum silicate Aluminum magnesium silicate Satd. picric acid + 10% NaOH Popular, inexpensive, rapid and easy to perform Requires automated equipment Elevated urea and creatinine in blood Decreased GFR but normal renal function Dehydration, shock, CHF Increased: BUN Normal: Creatinine True renal disease Decreased GFR Striking BUN level but slowly rising creatinine value BUN = >100 mg/dL Creatinine = >20 mg/dL Uric acid = >12 mg/dL Urinary tract obstruction Decreased GFR Nephrolithiasis, cancer or tumors of GUT Creatinine = normal or slightly increased Marked elevation of urea, accompanied by acidemia and electrolyte imbalance (K+ elevation) of renal failure Normocytic, normochromic anemia Uremic frost (dirty skin) Edema lec.mt 04 |Page | 24 Uric acid Hyperuricemia Hypouricemia Methods (Uric acid) Phosphotungstic acid mtd NaCN NaCO3 Lagphase Uricase method Para-amino hippurate test Phenolsulfonphthalein dye test Concentration tests Specific gravity Osmolality Direct methods (Osmolality) Incr. plasma osmolality Tubular failure Foul breath Urine-like sweat From purine (adenine and guanine) catabolism Excretion: 1g/day -Gout -Increased nuclear metabolism (leukemia, lymphoma, MM, polycythemia, hemolytic and megaloblastic anemia) – Tx: Allopurinol -Chronic renal disease -Lesch-Nyhan syndrome (HGPRT deficiency) Fanconi’s syndrome Wilson’s disease Hodgkin’s disease Stable for 3 days Potassium oxalate cannot be used Major interferences: Ascorbate and bilirubin Uric acid + Phosphotungstic acid ---(NaCN/NaCO3)--> Tungsten blue + Allantoin Folin Newton Brown Benedict Archibald Henry Caraway Incubation period after the addition of an alkali to inactivate non-uric acid reactants Simplest and most specific method Candidate reference method Uric acid (Absorbance at 293nm) ---[Uricase]--> Allantoin (No absorbance) Decrease in absorbance α uric acid concentration Measures renal plasma flow Reference method for tubular function Measures excretion of dye proportional to renal tubular mass 6 mg of PSP is administered IV Collecting tubules and loops of Henle Specimen: 1st morning urine Affected by solute number and mass SG >1.050: X-ray dye and mannitol 1.010 = SG of ultrafiltrate in Bowman’s space Total number solute particles present/kg of solvent (moles/kg solvent) Affectted only by number of solutes present Urine osmolality = due to urea Serum osmolality = due to sodium and chloride Det. by Colligative properties: Freezing point (incr. osm. = decr. FP) Vapor pressure (incr. osm. = decr. VP) Osmotic pressure (incr. osm. = incr. OP) Boiling point (incr. osm. = incr. BP) Freezing point osmometry = popular method Vapor pressure osmometry (Seebeck effect) Incr. vasopressin (H2O reabsorption) decr. plasma osmolality Increased: BUN, creatinine, calcium Decreased: Phosphate lec.mt 04 |Page | 25 Osmolal gap Osmolal gap: >12 mOsm/kg Normal Values (Kidney Function Tests) Liver Synthetic function Conjugation function Detoxification and Drug metabolism Excretory and Secretory functions Storage function Test measuring the Hepatic Synthetic Ability Test measuring Conjugation/Excretion Function Difference between measured and calculated osmolality Sensitive indicator of alcohol or drug overdose DKA Drug overdose Renal failure Creatinine Clearance: Male = 85-125 mL/min Female = 75-112 mL/min BUN = 8-23 mg/dL Creatinine = 0.5-1.5 mg/dL Uric acid: Male = 3.5-7.2 mg/dL Female = 2.6-6.0 mg/dL Renal plasma flow (PAH) = 600-700 mL/min Renal blood flow (PSP) = 1200 mL/min SG = 1.005-1.030 Osmolality: Serum = 275-295 mOsm/kg Urine (24-hr) = 300-900 mOsm/kg [<290 mOsm/kg = kidney damage] Urine osmolality: Serum osmolality = 1:1 to 3:1 [>1:1 = Glomerular disease] [1.2:1 = loss of renal concentrating ability] [<1:1 = Diabetes Insipidus] Liver Function Tests Receives 15 mL of blood per minute Lobule: anatomic unit Proteins, CHO, lipids, LPP, clotting factors, ketone bodies, enzymes Albumin: 12g/day Bilirubin metabolism Bilirubin: 200mg/day Drugs Ammonia Urea Excreted Bile acids: cholic acid and chenodeoxycholic acid Bile salts: bile acids + amino acids (glycine and taurine) Vitamins Glycogen Total Protein Determination: -Kjeldahl method -Biuret method -Folin-Ciocalteu (Lowry) method -UV absorption method -Electrophoresis -Refractometry -Turbidimetric and Nephelometric methods -Salt fractionation Prothrombin Time (Vitamin K Response Test) Bilirubin Assay: -Evelyn and Malloy method -Jendrassik and Grof Bromsulfonphthalein (BSP) Dye Excretion test lec.mt 04 |Page | 26 Test for Detoxification Function Plasma protein Kjeldahl (Digestion) mtd Biuret method Folin-Ciocalteu (Lowry) method Electrophoresis Gamma-spike Beta-gamma bridging Alpha2-globulin band spike Alpha1-globulin flat curve Alpha1, alpha2, betaglobulin band spikes Polyclonal gammopathy Small spikes in beta region Free hemoglobin Refractometry Turbidimetric and nephelometric methods Salt fractionation Albumin Enzyme tests: ALP, AST, ALT, 5’NT, GGT, OCT, LAP, LDH Ammonia: -Kjeldahl (Digestion) method -Nesslerization reaction -Berthelot reaction 0.2-0.4 g/dL higher than serum due to fibrinogen Standard reference method Measurement of nitrogen content Serum + Tungstic acid PFF 1g N2 = 6.54g protein 15.1-16.8% = N2 content of proteins Rgt: H2SO4 End product: NH3 Most widely used method (IFCC recommended) Req. at least 2 peptide bonds and an alkaline medium Rgts: Alkaline CuSO4 Rochelle salt (NaK Tartrate) NaOH KI End product: Violet color (545nm) Highest analytical sensitivity Oxidation of phenolic compounds (tyrosine, tryptophan, histidine) Rgts: Phenol (or phosphotungstic-molybdic acid) Biuret (color enhancer) End product: Blue color MI: elevated APRs (AAT, HPG, a1-x) Monoclonal gammopathy (multiple myeloma) In serum: Hepatic cirrhosis (IgA) In plasma: normal (fibrinogen) Nephrotic syndrome Juvenile cirrhosis (AAT deficiency) Inflammation Chronic inflammation (RA, malignancy) IDA (transferrin) “Blip” in the late alpha2 or early beta region Refractive index SSA TCA Salt: Sodium sulfate Soluble: Water Moderately concentrated salt solution Concentrated salt solution Insoluble: Hydrocarbon solvents Highly concentrated salt solution Saturated salt solution lec.mt 04 |Page | 27 Globulin Prothrombin time Albumin Hepatic cirrhosis Bromcresol green Bromcresol purple Other dyes for albumin Nephrotic syndrome Analbuminemia Bisalbuminemia Inverted A/G ratio Bilirubin Heme oxygenase Biliverdin reductase Urobilinogen Bilirubin 1 Bilirubin 2 Delta bilirubin Jaundice Pre-hepatic jaundice Hepatic jaundice Post-hepatic jaundice Soluble: Hydrocarbon solvents Weak salt solution Insoluble: Water Saturated salt solution Concentrated salt solution Differentiates intrahepatic disorder (prolonged PT) from extrahepatic obstructive liver disease (normal PT) Inversely proportional to the severity of the liver disease Low total protein + low albumin Most commonly used dye for albumin Most specific dye for albumin Hydroxyazobenzene benzoic acid (HABA) Methyl orange (MO) Albumin excretion: 20-30 g/day (-) albumin EP: 2 albumin bands Therapeutic drugs in serum Hepatic cirrhosis (IgA) Multiple Myeloma (IgG) Waldenström’s macroglobulinemia (IgM) Chronic inflammation Derived from hemoglobin myoglobin, catalase and cytochrome oxidase Protoporphyrin Biliverdin Biliverdin B1 Deconjugated bilirubin Non-polar bilirubin Free/Slow bilirubin Polar bilirubin One-minute/prompt bilirubin Regurgitative bilirubin Bilirubin tightly bound to albumin Delta bilirubin = TB-DB+IB Bilirubin >2 or 3 mg/dL Hemolytic B1 = increased B2 = normal UG = increased UB = negative Hepatocellular B1 = increased B2 = increased UG = increased UB = positive ALT = increased AST = increased Obstructive B1 = normal B2 = increased UG = decreased/negative UB = positive lec.mt 04 |Page | 28 Gilbert’s syndrome Crigler-Najjar syndrome Dubin-Johnson syndrome & Rotor syndrome Lucey-Driscoll syndrome Methods (Bilirubin) Van den Berg reaction Evelyn and Malloy method Jendrassik and Grof Bilirubin Rosenthal White method Mac Donald method Ammonia ALP = increased GGT = increased Cholesterol = increased Bilirubin transport deficit (uptake) B1 = increased B2 = decreased Conjugation deficit Type I = total UDPGT deficiency Type II = partial UDPGT deficiency B1 = increased B2 = decreased Danger: Kernicterus Bile is colorless Bilirubin excretion deficit Blockade of excretion into the canaliculi TB = increased B2 = increased Circulating inhibitor of bilirubin conjugation B1 = increased Free from hemolysis and lipemia Store in the dark Measured ASAP or w/in 2-3 hours Diazotization of bilirubin Accelerator: Methanol Diazo rgts: Diazo A (0.1% Sulfanilic acid + HCl) Diazo B (0.5% Sodium nitrite) Diazo blank (1.5% HCl) (+) pink to purple azobilirubin Affected by hemolysis Candidate reference method Accelerator: Caffeine sodium benzoate Buffer: Sodium acetate Ascorbic acid: terminates the initial reaction and destroys the excess diazo rgt Not falsely elevated by hemolysis Total bilirubin is measured 15 minutes after adding methanol or caffeine soln Absorbs light maximally at 450nm Double collection method Collection: -After 5 mins (50% dye retention) -After 30 mins (0% dye retention) Single collection method Collection: -After 45 mins (+/- 5% dye retention) From deamination of amino acids Elevated levels are neurotoxic and often associated w/ encephalopathy and acetaminophen poisoning Diagnosis of hepatic failure and Reye’s syndrome In severe liver disorder: NH3 circulation brain (conv. to glutamine) increases pH compromise the Kreb’s cycle Coma due to lack of ATP for the brain lec.mt 04 |Page | 29 Methods (Ammonia) Kjeldahl (Digestion) method Nesslerization of ammonia Berthelot reaction Normal Values (Liver Function Tests) Enzyme concentration Substrate concentration Saturation kinetics Cofactors Coenzymes Activators Metalloenzymes Inhibitors Competitive inhibitor Noncompetitive inhibitor Uncompetitive inhibitor Isoenzymes Specimen: Heparin or EDTA plasma Fasting is required Avoid smoking Prolonged standing of specimen: increased NH3 due to deamination Place on iced water immediately Avoid hemolysis Specimen PFF N2 ----------(hot conc. H2SO4 + CuSO4 + Hg + Selenium)----------> NH3 NH3 + K2Hg2I2 ----------(Gum Ghatti)----------> NH2Hg2I2 End color: Yellow (low to moderate N2) Orange brown (high N2) NH3 + Phenol + Hypochlorite -----(Na Nitroprusside)-----> Indophenol blue Total protein = 6.5-8.3 g/dL Albumin = 3.5-5.0 g/dL Globulin = 2.3-3.5 g/dL α1-globulin = 0.1-0.3 g/dL α2-globulin = 0.6-1.0 g/dL β-globulin = 0.7-1.1 g/dL γ-globulin = 0.8-1.6 g/dL Total bilirubin = 0.2-1.0 mg/dL Indirect bilirubin = 0.2-0.8 mg/dL Direct bilirubin = 0-0.2 mg/dL Urobilinogen: Urine = 0.1-1.0 Ehrlich units/2hrs (or 0.54 Ehrlich units/day) Stool = 75-275 Ehrlich units/100g feces (or 75-400 Ehrlich units/24hrs) Ammonia = 19-60 μg/dL Enzymes Serum Enzyme concentration = reaction rate Reagent If enzyme > substrate, substrate = reaction rate When substrate concentration reaches a maximal value, higher concentration of substrate no longer results in increased rate of reaction Nonprotein entities Organic compound Ex. NADP Coenzyme = Velocity Inorganic ions Alters spatial configuration of the enzyme for proper substrate binding Ex. Ca2+ (#1 activator), Zn2+ (LDH), Cl- (AMS), Mg2+ (CK, ALP) Inorganic ion attached to a molecule Ex. Catalase, cytochrome oxidase Interferes with the enzymatic reactions Binds to the active site of an enzyme Reversible (Substrate > Inhibitor) Binds to the allosteric site (cofactor site) Irreversible Binds to the enzyme-substrate complex Substrate = ES = Inhibition Same catalytic reactions but slightly different molecular structures Fractionation of isoenzymes lec.mt 04 |Page | 30 Temperature 40-50’C 60-65’C Temperature coefficient (Q10) pH Storage Hemolysis Lactescence or milky specimen Enzyme nomenclature Enzyme classification Oxidoreductases Transferases Hydrolases Lyases 37’C = optimum temperature for enzyme activity Temperature = Reaction rate (movement of molecules) Denaturation of enzymes Inactivation of enzymes For every 10OC increase in temperature, there will be a two-fold increase in enzyme activity Most physiologic reactions occur in the pH range of 7-8 Enzymes: -20’C = for longer period of time Substrate and Coenzymes: 2-8’C LDH (LD4 & 5): Room temperature Mostly increases enzyme concentration Decreases enzyme concentration 1st digit: classification 2nd and 3rd digits: subclass 4th digit(s): serial number “OTHLIL” Oxidoreductases Transferases Hydrolases Lyases Isomerases Ligases Redox reaction Dehydrogenases: -Cytochrome oxidase -LDH -MDH -Isocitrate dehydrogenase -G-6-PD Transfer of a chemical group other than hydrogen from 1 substrate to another Kinases, Transaminases, Aminotransferases: -CK -GGT -AST -ALT -OCT Hydrolysis/splitting by addition of water Esterases: -ACP -ALP -CHS -LPS Peptidases: -Trypsin -Pepsin -LAP Glycosidases: -AMS -Galactosidases Removal of groups w/o hydrolysis (product contains double bonds) Aldolase lec.mt 04 |Page | 31 Isomerases Ligases Active site Allosteric site Prosthetic group Holoenzyme Zymogen/proenzyme Emil Fisher’s/Lock and Key theory Kochland’s/Induced fit theory Enzyme kinetics Absolute specificity Group specificity Bond specificity Zero-order reaction First-order reaction Measurement of enzyme activity International Unit Katal Unit Nonkinetic assay Alkaline Phosphatase Phenylalanine L-leucine Levamisole 3M urea Methods (ALP) Decarboxylases: -Glutamate decarboxylase -Pyruvate decarboxylase -Tryptophan decarboxylase Intramolecular arrangements Glucose phosphate isomerase Ribose phosphate isomerase Joining of 2 substrate molecules Synthases Water-free cavity Where the substrate interacts Cavity other than the active site May bind regulatory molecules Coenzyme that is bound tightly to the enzyme Apoenzyme + Prosthetic group Inactive form of enzyme Shape of the key (substrate) must fit into the lock (enzyme) Based on the substrate binding to the active site of the enzyme Acceptable theory Enzymes catalyze reactions by lowering the activation energy level that the substrate must reach for the reaction to occur Enzyme combines w/ only 1 substrate and catalyzes only 1 reaction Enzymes combine w/ all the substrates in a chemical group Enzymes reacting w/ specific chemical bonds Reaction rate depends only on enzyme concentration Independent on substrate concentration Reaction rate is directly proportional to substrate concentration Independent on enzyme concentration Change in substrate concentration Change in product concentration Change in coenzyme concentration 1 micromole of substrate/minute 1 mole of substrate/second Absorbance is made at 10-second intervals for 100 seconds pH = 10.5 405nm Electrophoresis: (+) Liver Bone (Regan) Placenta Intestine (-) Heat fractionation: (Δ Stable) Regan Placenta Intestine Liver Bone (Δ Labile) Inhibits Regan, placental and intestinal ALP Inhibits Nagao ALP Inhibits liver and bone ALP Inhibits bone ALP Low temperature = Increased ALP 1. Bowers and McComb (PNPP) – IFCC recommended 2. Bessy, Lowry and Brock (PNPP) 3. Bodansky, Shinowara, Jones, Reinhart = BGP (beta glycerophosphate) 4. King and Armstrong = PP (phenylphosphate) 5. Klein, Babson & Read = Buffered PPP (phenolphthalein phosphate) 6. Huggins and Talalay = PPDP (phenolphthalein diphosphate) lec.mt 04 |Page | 32 7. Moss = ANP (alpha naphthol phosphate) Increased ALP Sprue Hyperparathyroidism Rickets (children) and osteomalacia (adults) Acid Phosphatase pH = 5.5 405nm Sources: Prostate (major), RBC, platelets, bone Prostatic ACP Inhibited by L-tartrate ions RBC ACP Inhibited by cupric and formaldehyde ions Methods (ACP) Room temperature (1-2 hrs) = decreased ACP Thymolphthalein monophosphate = specific substrate, substrate of choice (endpoint) Alpha-naphthyl phosphate = preferred for continuous monitoring methods 1. Gutman and Gutman = PP 2. Shinowara = PNPP 3. Babsonm Read and Phillips = ANP (continuous monitoring) 4. Roy and Hillman = Thymolphthalein monophosphate (endpoint) Aspartate Aminotransferase pH 7.5 (AST/SGOT) 340nm Sources: Cardiac tissue > Liver > Skeletal muscle > Kidney, pancreas, RBCs Alanine Aminotransferase pH 7.5 (ALT/SGPT) 340nm Major Source: Liver Methods (AST and ALT) 1. Karmen method = Kinetic 2. Reitman and Frankel = Endpoint -Color developer: DNPH -Color intensifier: 0.4N NaOH Increased Transaminases DeRitis ratio (ALT:AST) >1.0 = Acute hepatitis (Highest) 20x = viral or toxic hepatitis Moderate elevation = chronic hepatitis, hepatic cancer, IM Slight elevation = Hepatic cirrhosis, alcoholic hepatitis, obstructive jaundice Amylase Smallest enzyme (appears in urine) Earliest pancreatic marker P3: most predominant pancreatic AMS isoenzyme in AP Isoenzymes: S-type (ptyalin): anodal P-type (amylopsin): cathodal Methods (AMS) Samples w/ high activity of AMS should be diluted w/ NaCl to prev. inactivation Salivary AMS = inhibited by wheat germ lectin Substrate: Starch Saccharogenic Reducing sugars produced Classic reference method (SU) Amyloclastic Degradation of starch Chromogenic Increase in color intensity Coupled-enzyme Continuous-monitoring technique Lipase Late marker (AP) Most specific pancreatic marker Methods (LPS) Substrate: Olive oil/Triolein 1. Cherry Crandal (Reference method) 2. Tietz and Fiereck 3. Peroxidase coupling (most commonly used method) Lactate dehydrogenase Lacks specificity lec.mt 04 |Page | 33 Methods (LDH) 10-fold increase (LDH) 2-3x URL Creatine Kinase Duchenne’s muscular dystrophy CK-MB Methods (CK) Adenylate kinase N-acetylcysteine Liver cells and RBC Cleland’s reagent and glutathione Electrophoresis CK relative index (CKI) Aldolase 5’ Nucleotidase GGT Methods (GGT) Cholinesterase/ RBC: 150x LDH than in serum Sources: LD1 (α-HBD) and LD2 = Heart, RBC, Kidneys LD3 = pancreas, lungs, spleen LD4 an LD5 = liver and muscle LD6 = alcohol dehydrogenase 1. Wacker method (forward/direct) = pH 8.8, 340 nm, most commonly used 2. Wrobleuski LaDue (reverse/indirect) = pH 7.2, 2x faster 3. Wrobleuski Cabaud 4. Berger Broida Hepatic carcinoma and toxic hepatitis Viral hepatitis and cirrhosis Isoenzymes: CK-BB = most anodal, brain CK-MB = myocardium (20%) CK-MM = least anodal, skeletal and smooth muscles (Major, 94-100%) Total CK: 50x URL (highest) Most specific indicator of myocardial damage (AMI) Not elevated in angina 1. Tanzer-Gilbarg (forward/direct) = pH 9.0, 340nm 2. Oliver-Rosalki/ Rosalki & Hess (reverse/indirect) = most commonly used method, faster reaction; pH 6.8, 340nm Inside RBCs Interferes w/ CK assay Inhibited by adenosine monophosphate Activate CK Do not contain CK Partially restore lost activity of CK Reference method for CK CKI (%) = CK-MB/Total CK x 100 Isoenzymes: Aldolase A = Skeletal muscles Aldolase B = WBC, liver, kidney Aldolase C = brain tissue Marker for hepatobiliary diseases and infiltrative lesions of the liver Methods: 1. Dixon and Purdon 2. Campbell, Belfield and Goldberg Located in the canaliculi of the hepatic cells Differentates the source of an elevated ALP level Sensitive indicator of occult alcoholism Increased: Obstructive jaundice Alcoholic hepatitis (most sensitive) Substrate: gamma-glutamyl-p-nitroanilide 1. Szass 2. Rosalki and Tarrow 3. Orlowski Monitor effects of relaxants (succinylcholine) after surgery lec.mt 04 |Page | 34 Pseudocholinesterase Angiotensin-Converting Enzyme Ceruloplasmin Ornithine carbamoyl transferase G-6-PD Normal Values (Enzymes) Rise Peak Normalize Myoglobin 1-3 h 5-12 h 18-30 h Rise Peak Normalize Electroneutrality 40-75% ECF ICF Normal plasma Vasopressin deficiency Volume and Osmotic regulation Electrolytes Myocardial rhythm and Marker for organophosphate poisoning (Low CHS) Methods: 1. Ellman technic 2. Potentiometric A.k.a. peptidyldipeptidase A or Kininase II Converts angiotensin I angiotensin II (lungs) Indicator of neuronal dysfunction (Alzheimer’s disease – CSF) Ferrooxidase enzyme For hepatobiliary diseases Drug induced hemolytic anemia (primaquine, antimalarial drug) ALP = 30-90 U/L ACP: Total ACP (male) = 2.5-11.7 U/L Prostatic ACP = 0-3.5 ng/mL AST = 5-37 U/L ALT = 6-37 U/L AMS = 60-180 SU/dL (95-290 U/L) LPS = 0-1.0 U/mL LDH: Forward = 100-225 U/L Reverse = 80-280 U/L Acute Myocardial Infarction Markers Troponin T Troponin I CK-MB AST 3-4 h 3-6 h 4-8 h 6-8 h 10-24 h 12-18 h 12-24 h 24 h 7 d (10-14 d) 5-10 d 48-72 h 5d Acute Pancreatitis Markers Amylase Lipase 2-12 h 6h 24 h 24 h 3-5 d 7d Electrolytes Equal no. of cations and anions Balance of charges Average water content of the human body 1/3 of total body water 2/3 of total body water 93% water (Plasma: 13% > Whole blood) 7% solutes: (Increased in dehydration) -Proteins -Glucose -NPN -Lipids -Ions Excretion of 10-20L H2O everyday Sodium Potassium Chloride EC = Na+ > Cl- > HCO3- > Ca2+(5th) > iPO4 IC = K+ > Mg2+(4th) Potassium LD 12-24 h 48-72 h 10-14 d lec.mt 04 |Page | 35 contractility Neuromuscular excitability Cofactors (enzyme) ATPase ion pump Production and use of ATP from glucose Acid-base balance Replication of DNA and translation of mRNA Sodium Aldosterone Atrial natriuretic factor Hypernatremia Hyponatremia Thirst Pseudohyponatremia (artifactual) Methods (Na+) Potassium Specimen Considerations (K+) Hyperkalemia Hypokalemia Calcium Magnesium Calcium Magnesium (CK) Zinc Chloride (AMS) Potassium Magnesium Magnesium Phosphate Bicarbonate Magnesium Major contributor of osmolality (92%, together w/ Chloride and Bicarbonate) 100 mg/dL glucose = 1.6 mmol/L sodium Sodium Potassium = Magnesium Sodium Excess water loss Decreased water intake Hyperaldosteronism (Conn’s disease) Hypothalamic disease (Chronic hypernatremia) Renal failure SIADH (increased water retention) Marked hemolysis (dilutional effect) <125 mmol/L = severe neuropsychiatric symptoms Major defense against hyperosmolality and hypernatremia 1-2% water deficit = severe thirst 150-160 mEq/L Na+ = Moderate deficit of water >165 mEq/L Na+ = Severe water deficit Hyperlipidemia (turbidity) Hyperproteinemia 1. FEP 2. AAS 3. ISE = Glass aluminum silicate 4. Colorimetry = Albanese Lein Concentration in RBC is 105 mmol/L Reciprocal relationship with H+ 0.5% hemolysis = 0.5 mmol/L Gross hemolysis = 30% Serum K+ > Plasma K+ by 0.1-0.7 mmol/L because of platelets (clot) 10-20% in muscle activity 0.3-1.2 mmol/L = mild to moderate exercise 2-3 mmol/L = vigorous exercise; fist clenching Decreased resting membrane potential incr. contractility lack of muscle excitability Decreased renal excretion (Dehydration, renal failure, Addison’s disease) Acidosis (DM) Muscle injury Spironolactone Increased resting membrane potential arrhythmia lec.mt 04 |Page | 36 Vitamin D3 PTH Calcitonin Practical considerations (Ca2+) Leads to hypomagnesemia Vomiting Diuretics Cushing’s syndrome Alkalosis Insulin overdose pH by 0.1 = K+ by 0.2-1.7 mmol/L Lithium heparin plasma = preferred 1. FEP 2. AAS 3. ISE = Valinomycin gel 4. Colorimetry = Lockhead and Purcell Chief counter ion of sodium in ECF Chloride methods measure bromide and iodide Cl- = HCO31. Schales and Schales: -Mercurimetric titration -Diphenylcarbazone -Excess Hg++ -(+) Blue violet 2. Whiterhorn Titration method -Mercuric thiocyanate -Reddish complex 3. Ferric perchlorate 4. Cotlove chloridometer -Coulometric amperometric titration -Excess Ag++ 5. ISE -Ion exchange membrane -Tri-n-octylpropylammonium chloride decanol Renal tubular acidosis Metabolic acidosis Diabetes insipidus (Dehydration) Prolonged diarrhea Prolonged vomiting (HCl) Aldosterone deficiency (Na+ = Cl- = K+) Metabolic alkalosis (HCO3- = Cl-) Marked hemolysis (dilutional effect) 99% Bones 1% ECF Absorbed in the duodenum Absorption is favored at an acidic pH 50% = Free/Ionized/Unbound/Active Calcium 40% = Protein-bound (Albumin) 10% = Complexed with anions Ca2+ = absorption (intestine) and reabsorption (kidney) Ca2+ = resorption (bone) and reabsorption (kidney) Ca2+ = urinary excretion (major net loss of calcium) Serum = specimen of choice Albumin (1g/dL) = Ca2+ (0.8 mg/dL) Hypercalcemia Acidosis (Ca2+: from Bones Blood) pH and K+ Methods (K+) Chloride Specimen Considerations (Cl-) Methods (Cl-) Hyperchloremia Hypochloremia Calcium 3 Forms of Calcium lec.mt 04 |Page | 37 Hypocalcemia Primary hypocalcemia Secondary hypocalcemia Methods (Ca2+) Inorganic Phosphorus 3 Forms of Inorganic Phosphorus PTH Calcitonin Growth hormone Practical considerations Hyperphosphatemia Hypophosphatemia Methods (iPO4) Magnesium 3 Forms of Magnesium Cancer Hyperthyroidism Milk-alkali syndrome Tetany Alkalosis (Ca2+: from Blood Bones) Acute pancreatitis (Ca2+: binds to damage pancreatic tissues) Low PTH Parathyroid gland disease High PTH Renal failure ( excretion) 1. Clark Collip precipitation method -(+) Oxalic acid -Renal calculi 2. Ferro Ham Chloranilic acid precipitation method -(+)Chloranilic acid 3. Colorimetric = Ortho-Cresolphthalein complexone dyes -Dye: Arzeno III -8-hydroxyquinoline = chelates (inhibits) Mg2+ 4. EDTA titration method (Bachra, Dawer and Sobel) 5. AAS = Reference method 6. ISE = Liquid membrane 7. FEP 85% Bones 15% ECF (iPO4) Maximally absorbed in the jejunum (Ca2+: duodenum) Trancellular shift: Once absorbed inside cells, it no longer comes out used for energy production Dirunal variation: late morning, evening Organic phosphate = principal anion within cells Inorganic phosphate = part of the blood buffer (Measured in the clin.lab.) 55% = Free 35% = Complexed with ions 10% = Protein-bound PO4 = Ca2+ PO4 = Ca2+ PO4 (renal reabsorption) Fasting is required (Nonfasting: PO4) Hypoparathyroidism Renal failure Hypervitaminosis D Alcohol abuse = most common cause Primary hyperparathyroidism Avitaminosis D (Rickets, Osteomalacia) Most accurate: unreduced phosphomolybdate formation (340nm) 1. Fiske Subbarow Method (Ammonium molybdate method) -Reducing agents: Pictol, Elon, Senidine, Ascorbic acid -(+) Phosphomolybdenum blue 53% Bones 46% Muscles and soft tissues 1% Serum and RBC Vasodilator 55% = Free/Ionized/Physiologically active lec.mt 04 |Page | 38 PTH Aldosterone (& Thyroxine) Hypermagnesemia Hypomagnesemia Methods (Mg2+) Bicarbonate Chloride shift Anion Gap Increased AG Decreased AG Cystic Fibrosis (Mucoviscidosis) Pilocarpine Gibson & Cooke pilocarpine iontophoresis Iron Methods (Iron) Increased iron 30% = Protein-bound 10% = Complexed with ions Mg2+ = Ca2+ = PO4 Mg2+ = K+ = Na+ Addison’s disease Chronic renal failure Acute renal failure Chronic alcoholism 1. Calmagite -(+) Reddish-violet complex 2. Formazen dye method -(+) Colored complex 3. Magnesium Thymol blue method -(+) Colored complex 4. AAS = reference method 5. Dye-lake Method -Titan Yellow dye (Clayton Yellow or Thiazole yellow) 90% of the total CO2 HCO3- diffuses out of the cell in exchange for Cl- to maintain ionic charge neutrality w/in the cell Difference between unmeasured anions and unmeasured cations QC for ISE Uremia/renal failure Ketoacidosis Lactic acidosis Methanol poisoning Ethanol poisoning Ethylene glycol poisoning Salicylate poisoning Hypoalbuminemia Hypercalcemia Hyperlipidemia Multiple myeloma Defective gene: Cystic fibrosis transmembranous conductance regulator (Chromosome 7) Miconeum ileus (Infants) Foul-smelling stool URT infection Na+ and ClSweat inducer Reference method (Sweat sodium and chloride) Prooxidant 3-5g = Total body iron Ferrous = Hgb Ferric = Transferrin and Ferritin 1. Colorimetric = HCl and Ferrozine -(+) Blue color 2. Anodic stripping voltammetry Hemochromatosis lec.mt 04 |Page | 39 Decreased iron TIBC UIBC % Transferrin Saturation Transferrin Note Normal Values (Electrolytes) Viral hepatitis Non-IDA IDA Malnutrition Chronic infection UIBC + Serum Iron Increased: IDA, hepatitis, iron-supplemented pregnancy Decreased: Non-IDA, nephrosis TIBC – Serum iron Measure of reserve iron binding capacity of transferrin Index of iron storage Increased: Iron overdose, hemochromatosis, sideroblastic anemia Decreased: IDA (lowest), malignancy, chronic infection TIBC (μg/dL) x 0.70 = mg/dL Sodium 1/α Potassium Potassium 1/α Hydrogen ion Potassium α Magnesium Magnesium α Calcium Calcium 1/α Inorganic phosphate Chloride 1/α Bicarbonate Sodium: Serum = 135-145 mmol/L [Critical: 160 mmol/L and 120 mmol/L] CSF = 136-150 mmol/L Potassium: Serum = 3.5-5.2 mmol/L [Critical: 6.5 mmol/L and 2.5 mmol/L] Chloride: Serum = 98-107 mmol/L Sweat = 5-40 mmol/L [Critical: >65 mmol/L] Calcium: Total = 8.6-10 mg/dL (adult) and 8.8-10.8 mg/dL (child) Ionized = 4.6-5.3 mg/dL (adult) and 4.8-5.5 mg/dL (child) [Critical: <7.5 mg/dL] Inorganic Phosphate: Adult = 2.7-4.5 mg/dL Child = 4.5-5.5 mg/dL Magnesium: Serum = 1.2-2.1 mEq/L Anion Gap: w/ K+ = 10-20 mmol/L w/o K+ = 7-16 mmol/L Iron: Male = 50-160 μg/dL Female = 45-150 μg/dL TIBC: Adult = 245-425 μg/dL >40 y.o. = 10-250 μg/dL NB and Child = 100-200 μg/dL % Transferrin Saturation = 20-50% Blood Gases and pH lec.mt 04 |Page | 40 Regulation of Acid-Base balance 20:1 4:1 Expanded HendersonHasselbalch equation Chloride-isohydric shift pCO2 pO2 Metabolic Acidosis Metabolic Alkalosis Respiratory Acidosis Respiratory Alkalosis Full compensation Partial compensation Buffer base Methods for Blood Gases and pH Factors affecting Blood gases & pH measurements Methods Lungs and Kidneys CO2 + H2O <--(Carbonic anhydrase)--> H2CO3 H2CO3 <-------(Carbonic anhydrase)--> H+ + HCO3HCO3-: H2CO3 ratio HPO4: H2PO4 ratio pH = 6.1 + log [Total CO2 – (pCO2 x 0.03)] pCO2 x 0.03 Buffering effect of hemoglobin Index of efficiency of gas exchange Increased: Barbiturates, morphine, alcohol, heparin (12-15%) Reflects the availability of the gas in blood but not its content Excessive O2 supply acidosis Causes: -Bicarbonate deficiency -DKA (normochloremic acidosis) -Renal failure -Diarrhea (HCO3-) Compensation: Hyperventilation Compensated: HCO3- + pCO2 + pH <7.4 Causes: -Bicarbonate excess -Vomiting (Cl-) -Hypochloremia -Hypokalemia Compensation: Hypoventilation Compensated: HCO3- + pCO2 + pH >7.4 Causes: -CO2 excess (Hypoventilation) -COPD -Drug overdose (morphine, barbiturates, opiates) Compensation: Bicarbonate retention Compensated: HCO3- + pCO2 + pH <7.4 Causes -CO2 loss (Hyperventilation) Compensation: Bicarbonate excretion Compensated: HCO3- + pCO2 + pH >7.4 pH normal range pH near normal All forms of base that will titrate hydrogen ions Specimen: Arterial blood Blood gas analyzers: meas. pH, pCO2, pO2 For every 1OC above 37OC: pH by 0.015 pO2 by 7% pCO2 by 3% Bacterial contamination: consume O2 (pO2) Excess heparin (acid MPS) = pH Air exposure (bubbles): pO2 = 4 mmHg/2mins pCO2 = 4 mmHg/2mins 1. Gasometer lec.mt 04 |Page | 41 (Blood gases & pH) Whole blood total CO2 Transcutaneous electrodes Blood gas QC Normal Values (Blood gases and pH) Endocrine Paracrine Autocrine Juxtacrine Exocrine Neurocrine Neuroendocrine Glycoproteins Polypeptides Steroids Amines Hypothalamus Pineal gland Pituitary gland Anterior Pituitary a. Van Slyke b. Natelson -Mercury: produce vacuum -Caprylic alcohol: anti-foam reagent -Lactic acid -NaOH -NaHSO3 2. Electrodes a. pH = potentiometry -Silver-silver chloride electrode (Reference electrode) -Calomel electrode [Hg2Cl2] (Reference electrode) b. pCO2 = Severinghaus electrode (potentiometry) c. pO2 = Clark electrode (polarography-amperometry) Dissolved CO2 + H2CO3 + HCO3Continuous monitoring of pO2 Directly placed on the skin Min. requirement: -1 sample every 8 hours -3 levels of control (acidosis, normal, alkalosis) every 24 hours pH = 7.35-7.45 pCO2 = 35-45 mmHg Total CO2: WB arterial = 19-24 mmol/L WB venous = 22-26 mmol/L HCO3- = 21-28 mEq/L pO2 = 81-100 mmHg [Hypoxemia:] -Mild (61-80 mmHg) -Moderate (41-60 mmHg) -Severe (40 mmHg or less) O2 saturation = 94-100% Endocrinology Hormone blood circulation specific receptor Hormone interstitial space adjacent cell Hormone self-regulation Hormone direct cell-to-cell contact Hormone gut Hormone neurons extracellular space Hormone neurons nerve endings FSH, hCG, TSH, LH ACTH, ADH, GH, angiotensin, calcitonin, CCK, gastrin, glucagons, insulin, MSH, oxytocin, PTH, PRL, somatostatin Precursor: cholesterol Aldosterone, cortisol, estrogen, progesterone, testosterone, vitamin D Derived from amino acids Catecholamines, T3, T4 Connected to the posterior pituitary by the infundibulum stalk Hypophyseal hormones: TRH, GnRH, GH-IH, GH-RH, PIF Melatonin: decreases pigmentation of the skin Master Gland Located in the sella turcica or Turkish saddle True endocrine gland lec.mt 04 |Page | 42 (Adenohypophysis) GH (Somatotropin) Dwarfism Acromegaly GH deficiency tests Tests for Acromegaly FSH LH TSH (Thyrotropin) ACTH (Corticotropin) Prolactin Panhypopituitarism Pituitary ischemia (Shechan’s) Posterior pituitary (Neurohypophysis) Oxytocin ADH/AVP (Arginine vasopressin) Overnight water deprivation test (Conc. test) Neurogenic DI Nephrogenic DI SIADH Thyroid Gland Follicle Follicular cells Parafollicular or C cells Hormones: PRL, GH, FSH, LH, TSH, MSH, ACTH Most abundant of all pituitary hormones Structurally similar to PRL and HPL Markedly elevated during deep sleep Decreased GH Increased GH 1. Insulin tolerance test = Gold standard (Confirmatory test) 2. Arginine stimulation test = 2nd confirmatory test 1. Somatomedin C or insulin-like growth factor I (Screening) -Increased: Acromegaly -Decreased: GH deficiency 2. OGTT (Confirmatory) -75g glucose Spermatogenesis Helps Leydig cells to produce testosterone (male) Ovulation (female) Synthesis of androgens, estrogens, and progesterone Stimulates thyroid gland to produce T3 and T4 Increased: 1’ hypothyroidism, 2’ hyperthyroidism Decreased: 1’ hyperthyroidism, 2’ hypothyroidism, 3’ hypothyroidism Highest: 6-8 AM Lowest: 6-11 PM Not allowed to have contact with glass because it adheres to glass surface Collect blood in plastic tubes Initiation and maintenance of lactation Inhibited by Dopamine Highest: 4AM and 8AM, and 8PM and 10PM Increased: Menstrual irregularity, infertility, amenorrhea, galactorrhea From pituitary tumor (adenoma) or Ischemia Hemorrhage or shock in a pregnant female at the time of deliver Release but not produce oxytocin and vasopressin Uterine contraction and milk ejection H2O reabsorption (DCT and CD) Stimulus: Increased plasma osmolality (>295 mOsm/kg), decreased blood vol. Promotes factor VII and vWF release Diagnostic test for ADH True Diabetes Insipidus Failure of the pituitary gland to secrete ADH Failure of the kidneys to respond to normal or elevated ADH Syndrome of inappropriate ADH Sustained production of ADH Decreased urine volume Low plasma osmolality Low serum electrolytes Butterfly-shaped 2 lobes = connected by the isthmus Fundamental structural unit of the thyroid gland Secrete T3 and T4 Secrete calcitonin lec.mt 04 |Page | 43 Thyroglobulin Thyroid hormone Biosynthesis Protein-bound hormones Free hormones (FT3/FT4) Reverse T3 (rT3) I2 intake <50 μg/day T3 T4 TBG TBPA (Transthyretin) TBA Thyroid autoantigens Thyroid disorders Primary hyperthyroidism Secondary hyperthyroidism T3 Thyrotoxicosis (Plummer’s disease) Graves’ disease (Diffuse toxic goiter) Riedel’s thyroiditis Subclinical hyperthyroidism Subacute granulomatous/ Subacute nonsuppurative/ De Quervain’s thyroditis Hypothyroidism Preformed matrix containing tyrosyl groups Stored in the follicular colloid of the thyroid gland 1. Trapping of Iodine 2. Iodination: I2 Tyrosine ring MIT and DIT 3. Condensation: MIT+DIT=T3 / DIT+DIT=T4 4. Release: T3/T4 Blood circulation 5. Transport of T3/T4 by proteins Metabolically inactive Biologically inert Do not enter cells Storage sites Physiologically active Readily enters cells From removal of one iodine from T4 (product of T4 metabolism) Metabolically inactive Deficiency of hormone secretion 3,5,3’-Triiodothyronine Most active thyroid hormonal activity 75-80% is produced from the tissue deiodination of T4 Diagnosis of T3 thyrotoxicosis 3,5,3’5’-Tetraiodothyronine Principal secretory product All originated in the thyroid gland Transports 70-75 of TT4, and majority of T3 Transports 15-20% of TT4 No affinity for T3 Transports T3 and 10% of T4 TPO Tg TSHR Screening is recommended when a person reaches 35 yrs old and every 5 yrs thereafter T3 and T4 TSH T3 and T4 TSH T3 N-T4 TSH 1’ Hyperthyroidism Most common cause of thyrotoxicosis (autoimmune) Women > Men Anti-TSH receptor Thyroid woody or stony-hard mass No symptoms N-T3 and T4 TSH Hyperthyroidism Painful thyroiditis Neck pain, low-grade fever (-) anti-TPO, ESR and Tg Treatment: Levothyroxine lec.mt 04 |Page | 44 Primary hypothyroidism Hashimoto’s disease (Chronic autoimmune thyroiditis) Myxedema coma Secondary hypothyroidism Tertiary hypothyroidism Congenital hypothyroidism (Cretinism) Subclinical hypothyroidism TRH stimulation test Radioactive Iodine Uptake (RAIU) Thyroglobulin (Tg) assay rT3 Free Thyroxine Index (FT4I) TT3, FT3, FT4 T3 Uptake test TBG test Fine-needle aspiration Recombinant Human TSH Tanned Erythrocyte Hemagglutination method Serum calcitonin test T3 and T4 TSH Most common cause of 1’ hypothyroidism Thyroid is replaced by a nest of lymphoid tissue (T cells) Goiter (+) anti-TPO TSH Severe form of 1’ hypothyroidism Peculiar nonpitting swelling of the skin Skin is infiltrated by mucopolysaccharides “Puffy” face, thin eyebrows T3 and T4 TSH T3 and T4 TSH TRH Mental retardation (child) Screening: T4 Confirmatory: TSH N-T3 and T4 TSH Most specific and sensitive test for diagnosing thyroid disease Confirm borderline cases and euthyroid Graves’ disease : 1’ hypothyroidism : Hyperthyroidism Measure the ability of the thyroid gland to trap iodine Postoperative marker of thyroid cancer : Untreated and metastatic differentiated thyroid cancer, hyperthyroidism : Hypothyroidism, thyrotoxicosis factitia Assess borderline or conflicting laboratory results Indirectly assesses the level of FT4 in blood Equilibrium relationship of bound T4 and FT4 Reference method: Equilibrium dialysis FT4I = TT4 x T3U(%) or TT4 x THBR 100 FT4 test: differentiates drug induced TSH elevation and hypothyroidism TT3 or FT3: confirm hyperthyroidism Reference method (FT4): Equilibrium dialysis Measures the number of available binding sites of the thyroxine binding proteins (TBG) TBG = T3U TBG = T3U Confirm results of FT3 or FT4 or abnormalities in the relationship of TT4 and THBR test Estrogen: TBG Androgen: TBG Most accurate tool in the evaluation of thyroid nodules Test patients w/ thyroid cancers for the presence of residual or recurrent dis. Test for anti-Tg disorders Marker for familial medullary thyroid carcinoma lec.mt 04 |Page | 45 FT4 and TSH FT3 and FT4 Euthyroid sick syndrome Parathyroid gland PTH 1’ hyperparathyroidism 2’ hyperparathyroidism 3’ hyperparathyroidism Hypoparathyroidism Hyperparathyroidism Hypoparathyroidism Adrenal glands Adrenal cortex CPPP ring 3 layers (Adrenal cortex) Cortisol Porter-Silber method Best indicators of thyroid status More specific indicators of thyroid function than meas. of total hormone Not affected by TBG Acutely ill but without thyroid disease T3 and T4 N/ TSH rT3 4 parathyroid glands Smalles endocrine gland Hypercalcemic hormone Ca2+ (bone resorption and renal reabsorption) and Mg2+ iPO4 Defective: Parathyroid gland Most common cause of hypercalcemia Parathyroid adenoma PTH and iCa2+ Hypercalciuria Phosphaturia Hypophosphatemia If goes undetected severe demineralization (osteitis fibrosa cystica) In response to Ca2+ Hyperplasia of all 4 glands Causes: Vit. D deficiency and chronic renal failure PTH Ca2+ Occurs w/ 2’ hyperparathyroidism ( Ca2+) Autonomous function of hyperplastic PT glands or PT adenoma PO4 Calcium phosphates precipitate in soft tissues Accidental injury of the PT glands (neck) during surgery Autoimmune parathyroid destruction PTH = Ca2+ Acidosis Alkalosis Pyramid-shaped Above the kidneys Adrenal cortex = outer (yellow) Adrenal medulla = inner (dark mahogany) Has prime effects on blood pressure Major site of steroid hormone production G cells: convert cholesterol pregnenolone 17-carbon skeleton derived from cholesterol 1. Zona Glomerulosa = Mineralocorticoids (Aldosterone) 2. Zona Fasciculata = Glucocorticoids (Cortisol) 3. Zona Reticularis = Weak androgens (androstenedione, DHEA) Gluconeogenesis hyperglycemia The only adrenal hormone that inhibit the secretion of ACTH Anti-inflammatory and immunosuppressive Diurnal: 6-8AM / 10PM-12AM Urinary metabolites: 17-OHCS and 17-KGS Meas. 17-OHCS lec.mt 04 |Page | 46 Zimmerman reaction Pisano method Kober reaction Cushing’s syndrome (Hypercortisolism) Screening tests (Cushing’s) Confirmatory tests (Cushing’s) Addison’s disease (1’ Hypocorticolism) 2’ Hypocorticolism ACTH Stimulation test (Corsyntropin stimulation test) Metyrapone test 24-hour urine free cortisol HPLC-MS ITT (Insulin tolerance test) Serum ACTH ACTH Congenital Adrenal Rgt: DNPH in H2SO4 + Alcohol (+) Yellow Meas. 17-KGS Rgt: m-dinitrobenzene (+) Reddish purple Oxidation procedure: Norymberski (Na+ bismuthate) For quantitating metanephrines and normetanephrines For estrogen Rgt: H2SO4 + hydroquinone (+) Reddish brown color Excessive production of cortisol and ACTH Overuse of corticosteroids Buffalo hump Hyperglycemia Hypertension Hypercholesterolemia Lymphocytes 1. 24-hour urine free cortisol test 2. Overnight dexamethasone suppression tests = Most widely used (1mg) 3. Salivary cortisol test 1. Low-dose dexamethasone suppression test (0.5mg) 2. Midnight plasma cortisol 3. CRH stimulation test Primary adrenal insufficiency Cortisol and aldosterone ACTH (+) Hyperpigmentation Screen: ACTH Stimulation Test Secondary adrenal insufficiency Hypothalamic-pituitary insufficiency ACTH Test: ACTH Stimulation test Corsyntropin: synthetic coritsol and aldosterone stimulator Differentiates: 2’ adrenal insufficiency (ACTH) from 3’ adrenal insufficiency ( ACTH) Metyrapone: inhibitor of 11 β-hydroxylase Measures the ability of the pituitary gland to respond to declining levels of circulating cortisol, thereby secrete ACTH Alternative diagnostic or confirmatory test for 2’ or 3’ adrenal insufficiency (+): ACTH Most sensitive and specific screening test for excess cortisol production because plasma cortisol is affected by diurnal variation Methods: HPLC or GC-MS Reference method for measuring urinary free cortisol Gold standard for 2’ and 3’ hypocorticolism Confirms borderline response to ACTH stimulation test Differentiates: Cushing’s disease (ACTH) Cushing’s syndrome (0-ACTH) 17-OHCS and 17-KS Enzyme deficiencies: lec.mt 04 |Page | 47 Hyperplasia Aldosterone (Aldo) Conn’s disease (1’ hyperaldosteronism) 2’ Hyperaldosteronism Liddle’s syndrome Bartter’s syndrome Gitelman’s syndrome Hypoaldosteronism Postural stimulation test Florinef Weak androgens DHEA (Dehydroepiandrosterone) Adrenal medulla 9:1 Norepinephrine Epinephrine 1.) 21-hydroxylase = most common 2.) 11 β-hydroxylase = 2nd most common 3.) 3β-hydroxysteroid dehydrogenase-isomerase 4.) C-17,20-lyase/17α-hydroxylase Cortisol ACTH Androgens (hirsutism, virilization, amenorrhea, pseudohermaphroditism) Electro-regulating hormone Na+ and Cl K+ and H+ at night 18-hydroxysteroid dehydrogenase: enzyme needed for aldosterone synthesis Aldosterone-secreting adrenal adenoma Screen: Plasma Aldo conc./Plasma renin activity ratio (PAC/PRA ratio) -(+): >50 ratio Confirm: Saline suppression test -(+): >5 ng/dL aldosterone Excessive production of renin Pseudohyperaldosteronism Resembles 1’ aldosteronism clinically Aldosterone (-) Hypertension Bumetanide-sensitive chloride channel mutation Aldosterone and Renin Thiazide-sensitive transporter mutation Aldosterone Destruction of the adrenal glands Glucocorticoid deficiency 21-hydroxylase deficiency Test for aldosterone Synthetic mineralocorticoid Precursors for the production of more potent androgens and estrogens Precursors: Pregnenolone and 17-OH pregnenolone Examples: DHEA and androstenedione Bound to steroid hormone binding globulin (SHBG) : Virilization (pseudohermaphroditism) Principal adrenal androgen Converted to estrone Chromaffin cells: secrete catecholamines Precursor: L-tyrosine Norepinephrine/Epinephrine ---(Monoamine oxidase and Catechol-0-methyltransferase)---> Metanephrines and VMA Norepinephrine: Epinephrine ratio Primary amine in CNS Metabolites: -3-methoxy-4-hydroxyphenylglycol (MHPG) = Major metabolite -VMA Secondary amine Most abundant medullary hormone “Flight or fight hormone” Metabolites: lec.mt 04 |Page | 48 Dopamine Pheochromocytoma Clonidine test Neuroblastoma Methods (Catecholamines) Estrogens Markers for Down Syndrome Karyotyping or FISH typing Progesterone Tests for menstrual cycle dysfunction and anovulation Tests for female infertility Pancreas (Exocrine) Pancreas (Endocrine) hCG Human placental lactogen (HPL) -Vanillylmandelic acid (VMA) = Major metabolite -Metanephrines -Normetanephrines -HVA Primary amine From the decarboxylation of 3,4-Dihydroxyphenylalanine (DOPA) Major metabolite: Homovanillic acid (HVA) Tumors of the adrenal medulla Catecholamines Classic “Spells”: tachycardia, headache, chest tightness, sweating, hypertension Differentiates: Pheochromocytoma (Catecholamines not suppressed) from Neurogenic hypertension (50% decreased in catecholamines) Norepinephrine (Children) urinary HVA, VMA or both and dopamine Specimen: 24-hr urine and plasma 1. Chromatography: HPLC or GC-MS 2. RIA: sensitive screening test ->2000pg/mL = diagnostic for pheochromocytoma Estrone = Postmenopausal women Estradiol = Premenopausal women (most potent, secreted by the ovary) Estriol = Pregnancy (placenta) AFP Unconjugated Estriol hCG Inhibin A Test for Down syndrome (amniotic fluid) Produced mainly by the corpus luteum Det. whether ovulation has occurred Luteal phase Estrogen Progesterone FSH LH hCG PRL FT4 TSH FSH LH Estradiol Progesterone Digestive enzymes (AMS, LPS) Acinus: functional secretory unit Hormones: Alpha cells (20-30%) = glucagon Beta cells (60-70%) = insulin Delta cells (2-8%) = somatostatin Produced by the syncytiotrophoblasts (placenta) Maintain progesterone production by the corpus luteum Stimulates development of mammary gland Increases maternal plasma glucose levels lec.mt 04 |Page | 49 Diagnosis of intrauterine growth retardation Gastrin Serotonin (5-hydroxytryptamine) 5-HIAA Somatostatin 1’ amenorrhea 2’ amenorrhea Cushing’s disease Cushing’s syndrome Gynecomastia Hirsutism Mullerian agenesis Nonthyroidal illness Sipples syndrome (MEN II) Stein-Leventhal syndrome Thyroid stones Normal Values (Endocrinology) Mixed function oxidase (MFO) system Intravenous route Liberation Absorption Distribution Metabolism Excretion Bioavailable fraction (f) Vd of a drug First-pass hepatic Secreted by G cells (stomach) Stimulates parietal cells to secrete HCl Stimulus: Amino acid Zollinger-Ellison syndrome Pernicious anemia Synthesized by argentaffin cells (GIT) Metabolite: 5-HIAA Diagnostic marker for carcinoid syndrome Test: Ehrlich’s aldehyde test = (+) purple color A.k.a. GH-IH Inhibitor of GH, glucagon and insulin Menstruation having never occurred Absence of menses for 6 months Abnormal increased secretion of ACTH Chronic excessive production of cortisol by the adrenal cortex -Large doses of glucocorticoids -Pituitary tumor (ACTH) = most common cause Development of breast tissue in males Excessive hair growth w/ a male distribution pattern in a female Most common endocrine disorder in women Congenital malformation or absence of the fallopian tubes, uterus or vagina N-FSH, LH and testosterone Illness that do not directly involve the thyroid gland Medullary carcinoma of the thyroid Pheochromocytoma Parathyroid adenoma Mild hirsutism w/ normal menses to excessive hirsutism w/ amenorrhea A.k.a. thyroid crisis Life-threatening Uncontrolled thyrotoxicosis T3: Adult = 80-200 ng/dL Children 1-14 y.o. = 105-215 ng/dL T4: Adult = 5.5-12.5 μg/dL Neonate = 11.8-22.6 μg/dL T3U = 25-35% Therapeutic Drug Monitoring Biochemical pathway responsible for the greatest portion of drug metabolism 100% bioavailability Drug Release Drug Blood (most: by passive diffusion) Drug Tissues Drug Chemical modification Drug metabolites excreted Fraction of the dose that reaches the blood Dilution of the drug after it has been distributed in the body Drugs Liver Decreased bioavailability lec.mt 04 |Page | 50 metabolism First order elimination Pharmacodynamics Pharmacokinetics Pharmacogenomics Therapeutic index Trough concentration Peak concentration Cardioactive Drugs Class I Class II Class III Class IV Digoxin Lidocaine (Xylocaine) Quinidine Procainamide (Pronestyl) Disopyramide Propanolol Amiodarone (Cordarone) Verapamil Antibiotics Aminoglycosides Vancomycin Antiepileptic Drugs Phenobarbital Phenytoin (Dilantin) Valproic acid (Depakene) Carbamazepine (Tegretol) Ethosuximide (Zarontin) Gabapentin (Neurontin) Others (Antiepileptic) Psychoactive Drugs Lithium Tricyclic antidepressantas (TCA) Linear relationship bet. the amt. of drug eliminated per hour and the blood level of drug Relationship bet. drug concentration at the target site and response of the tissues Relationship bet. drug dose and drug blood level Study of genes that affect the performance of a drug in an individual Ratio bet. the minimum toxic and maximum therapeutic serum conc. Lowest concentration of a drug obtained in the dosing interval Drawn immediately (or 30 mins) before the next dose Highest concentration of a drug obtained in the dosing interval Drawn one hour after an orally administered dose (except digoxin) Rapid Na+ channel blockers (Procainamide, Lidocaine, Quinidine) Beta receptor blockers (Propanolol) K+ channel blockers (Amiodarone) Ca2+ channel blockers (Verapamil) Tx: CHF Local anesthetic 1’ product of hepatic metabolism: MEGX (monoethylglycinexylidide) Common formulations: Quinidine sulfate and Quinidine gluconate Hepatic metabolite: NAPA (N-acetylprocainamide) Toxic effect: reversible lupus-like syndrome Substitute for quinidine Anticholinergic effects Tx: angina pectoris Iodine-containing drug Tx: angina, hypertension, supraventricular arrhythmias Tx: Gram (-) bacterial infections Nephrotoxic and ototoxic Tx: Gram (+) cocci and bacilli Toxic effects: “Red man syndrome” Nephrotoxic and ototoxic Long acting barbiturate Enhances bilirubin metabolism Inactive proform: Primidone Injectable proform: fosphenytoin Tx: petit mal and grand mal Tx: grand mal Drug of choice for controlling petit mal seizure Similar to neurotransmitter GABA Topiramate Lamotrigine (Lamictal) Felbamate Tx: Bipolar disorders (Manic depression) Imipramine Amitriptyline Doxepin lec.mt 04 |Page | 51 Fluoxetine (Prozac) Nortriptyline Tradazone Major metabolite: Desipramine Blocks reuptake of serotonin Tx: Obsessive-compulsive disorders Bronchodilator Theophylline Tx: Asthma and other COPD Anti-inflammatory and Analgesic Drugs Salicylates/Aspirin Antiplatelet (inhibits cyclooxygenase) (Acetylsalicylic acid) Method: Trinder assay Acetaminophen (Tylenol) Hepatotoxic Ibuprofen Lower risk of toxicity than salicylates and acetaminophen Neuroleptics (Antipsychotic major tranquilizers) Neuroleptics Block the action of dopamine and serotonin Tx: Schizophrenia 2 classes: -Phenothiazines (chlorpromazine) -Butyrophenones (haloperidol) Examples: -Risperdal -Olonzapine (Zyprexa) -Quetiapine (Seroquel) -Aripiprazole (Abilify) Immunosuppressants Cyclosporine Tacrolimus (FK-506) Rapamycin (Sirolimus) Mycophenolate mofetil Lefluamide Chemotherapeutic agents Busulfan Methotrexate Toxicology Toxic Agents Alcohols (%w/v) Common CNS depressants 0.01-0.05 No obvious impairment, some changes observable on performance testing 0.03-0.12 Mild euphoria, decr. inhibitions, some impairment of motor skills 0.09-0.25 Decr. inhibitions, loss of critical judgment, memory impairment, decr. rxn time 0.18-0.30 Mental confusion, dizziness, strongly impaired motor skills (slurred speech) 0.27-0.40 Unable to stand/walk, vomiting, impaired consciousness 0.35-0.50 Coma and possible death ≥0.10 Presumptive evidence of driving under influence of alcohol Ethanol (Grain alcohol) Most common abused drug Ethanol Acetic acid Major metabolic pathway: Ethanol ------(Alcohol Dehydrogenase)------> Acetaldehyde Testing: Use benzalkonium chloride as antiseptic Methanol (Wood alcohol) Cause blindness Methanol Formaldehyde Formic acid (liver) Isopropanol Liver metabolism: (Rubbing alcohol) Isopropanol Acetone Ethylene glycol Antifreezing agent (1,2-ethanediol) Ethylene glycol Oxalic acid and glycolic acid (+) Monohydrate calcium oxalate crystals lec.mt 04 |Page | 52 Carbon Monoxide Cyanide Arsenic Cadmium Lead Mercury Drugs of Abuse Opiates Tranquilizers Barbiturates: Sedative Hypnotics Dopaminergic pathway stimulants Hallucinogens Amphetamines Annabolic steroids Cannabinoids Tetrahydrocannabinol (THC) Cocaine (Crack) Colorless, odorless, tasteless gas Has 210x greater affinity than O2 for Hgb “Cherry-red” color of the face and blood Specimen: EDTA whole blood Method: Co-oximetry (HbCO measurement) Binds to iron (ferric and ferrous) containing substances like hemoglobin and cytochrome oxidase “Odor of bitter almonds” Antidote: Sodium thiosulfate, amyl and sodium nitrite “Odor of garlic” “Metallic taste” Hair and nails: “Mees lines” Method: Reinsch test (Flat black) Significant environmental pollutant (+) GGT in urine sample Blocks D-ALA synthase and Ferrocheletase “Wrist drop or Foot drop” manifestation Tx: EDTA and dimercaptosuccinic acid (DMA) – remove lead Free erythrocyte protoporphyrin (+) Basophilic stippling (course) Amalgamate: mix or merge w/ other substances Specimen: -Whole blood (organic mercury) -Urine (inorganic mercury) Method: Reinsch test (Silvery gray) Morphine Codeine Heroin Methadone Diazepam (Valium) Oxazepam Phenobarbital Pentobarbital Amobarbital Cocaine Benzoylecgonine Amphetamine Phencyclidine Lysergic acid diethylamide Tetrahydrocannabinol Methaqualone Increase mental alertness (“Uppers”) MDMA (methylenedioxymethamphetamine) = ecstasy Methamphetamine HCl = shabu Improves athletic performance by increasing muscle mass Marijuana and hashish Psycoactive substance of marijuana Urinary metabolite: 11-nor-deltatetrahydrocannabinol (THC-COOH) Alkaloid salt Admin: Insufflation of IV or by inhalation/snorting Derived from coca plant (erythroxylon) lec.mt 04 |Page | 53 Opiates Phencyclidine (Angel dust or angel hair) Sedative hypnotics Lysergic acid diethylamide (LSD, Lysergide) Methaqualone (Quaalude) Vitamins Vitamin A Vitamin E Vitamin D2 Vitamin D3 Vitamin K Vitamin B1 Vitamin B2 Vitamin B3 Vitamin B5 Vitamin B6 Vitamin B9 Vitamin B12 Vitamin C Biotin Carnitine Cardiac toxicity Prozac: inhibit the action of cocaine Urine metabolite: benzoylecgonine From opium poppy Heroin Morphine Codeine Methadone Major metabolites: N-acetylmorphine (heroin) and morphine Antagonist: Nalaxone (Narcan) Hallucinogen Admin: Ingestion or inhalation Major metabolite: Phencyclidine HCl Barbiturates (Secobarbital, pentobarbital, Phenobarbital) Benzodiazopines: Diazepam (Valium), Lorazepam (Ativan), Chlordiazepoxide (Librium) Major metabolite (barbiturates): Secobarbial “Undulating vision” “Bad trip” – panic reactions Pyramidal signs (Hypertonicity, hyperreflexia, myoclonus) Vitamins Water soluble: B1, B2, B3, B5, B6, B9, B12, Biotin, C, Carnitine Fat soluble: A, D, E, K CN: Retinol Def: Night blindness CN: Tocopherol Def: Mild hemolytic anemia, RBC fragility CN: Ergocalciferol, Cholecalciferol (D2), 1,25-dihydroxycholecalciferol (D3) Def: Rickets (young), Osteomalacia (adult) CN: Phylloquinones, Menaquinones Def: Hemorrhage CN: Thiamine Def: Beriberi, Wernicke-Korsakoff syndrome CN: Riboflavin Def: Angular stomatitis, dermatitis, photophobia CN: Niacin/Niacinamide/Nicotinic acid/Nicotinamide Def: Pellagra (dermatitis, disorientation, weight loss) CN: Panthotenic acid Def: Depressed immune system, muscle weakness CN: Pyridoxine, Pyridoxal Def: Facial seborrhea CN: Folic acid, Pteroylglutamic acid Def: Megaloblastic anemia CN: Cyanocobalamin Def: Megaloblastic anemia, neurologic abnormalities CN: Ascorbic acid Def: Scurvy Def: Dermatitis Def: Muscle weakness, fatigue lec.mt 04 |Page | 54
